Compounds, methods and pharmaceutical compositions for inhibiting PARP

ABSTRACT

The present invention provides compounds which inhibit poly(ADP-ribose) polymerase (“PARP”), compositions containing these compounds and methods for using these PARP inhibitors to treat, prevent and/or ameliorate the effects of the conditions described herein.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of application Ser. No. 11/834,334,filed on Aug. 6, 2007 now U.S. Pat. No. 7,456,178. Application Ser. No.11/834,334 is a divisional of application Ser. No. 10/853,714, filed onMay 26, 2004 now U.S. Pat. No. 7,268,138, and for which claims domesticpriority benefits under 35 U.S.C. §120 from, PCT/US04/016524, filed May,26, 2004, and this application claims priority benefit under 35 U.S.C.§119(e) from, Application No. 60/473,475, filed May 28, 2003. The entirecontent of each of these applications are hereby incorporated byreference.

The present invention provides compounds, methods and pharmaceuticalcompositions for inhibiting the nuclear enzyme poly(adenosine5′-diphospho-ribose) polymerase [“poly(ADP-ribose) polymerase” or“PARP”, which is also referred to as ADPRT (NAD:protein (ADP-ribosyltransferase (polymersing)), pADPRT (poly(ADP-ribose) transferase) andPARS (poly(ADP-ribose) synthetase). Moreover, the present inventionprovides methods of using PARP inhibitors of the invention to preventand/or treat tissue damage resulting from cell damage or death due tonecrosis or apoptosis; neural tissue damage resulting from, for example,ischemia and reperfusion injury, such as cerebral ischemic stroke, headtrauma or spinal cord injury; neurological disorders andneurodegenerative diseases, such as, for example, Parkinson's orAlzheimer's diseases and multiple sclerosis; to prevent or treatvascular stroke; to treat or prevent cardiovascular disorders, such as,for example, myocardial infarction; to treat other conditions and/ordisorders such as, for example, age-related muscular degeneration, AIDSand other immune senescence diseases, arthritis, atherosclerosis, ataxiatelangiectasia, cachexia, cancer, degenerative diseases of skeletalmuscle involving replicative senescence, diabetes (such as diabetesmellitus), inflammatory bowel disorders (such as colitis and Crohn'sdisease), acute pancreatitis, mucositis, hemorrhagic shock, splanchnicartery occlusion shock, multiple organ failure (such as involving any ofthe kidney, liver, renal, pulmonary, retinal, pancreatic and/or skeletalmuscle systems), acute autoimmune thyroiditis, muscular dystrophy,osteoarthritis, osteoporosis, chronic and acute pain (such asneuropathic pain), renal failure, retinal ischemia, septic shock (suchas endotoxic shock), local and/or remote endothelial cell dysfunction(such are recognized by endo-dependent relaxant responses andup-regulation of adhesion molecules), inflammation and skin aging; toextend the lifespan and proliferative capacity of cells, such as, forexample, as general mediators in the generation of oxidants,proinflammatory mediators and/or cytokines, and general mediators ofleukocyte infiltration, calcium ion overload, phospholipid peroxidaion,impaired nitric oxide metabolism and/or reduced ATP production; to altergene expression of senescent cells; or to radiosensitize hypoxic tumorcells.

Some of the PARP inhibitors used in the inventive methods andpharmaceutical compositions can be readily prepared by standardtechniques of organic chemistry, utilizing the general syntheticpathways and examples depicted in publications Wu et al, The ProtectiveEffect of GPI 18078, a Novel Water Soluble Poly (ADP-Ribose) PolymeraseInhibitor in Myocardial Ischemia-Reprefusion Injury, ExperimentalBiology 2003 (FASEB), Apr. 11-15, 2003; Wu et al, Myocardial Protectionand Anti-Inflammatory Effect of GPI 15427, a Novel Water Soluble Poly(ADP-Ribose) Polymerase Inhibitor: Comparison with GPI 6150,Experimental Biology 2003 (FASEB), Apr. 11-15, 2003; Kalish et al,Design, Synthesis and SAR of PARP-1 Inhibitors, ISMC Meeting, Barcelona,Sep. 4, 2002; Xu et al, Design and Synthesis of Novel Potent Poly(ADP-Ribose) Polymerase (PARP) Inhibitors, 224^(th) ACS NationalMeeting, Boston, Aug. 18-23, 2002; Williams et al, Intravenous Deliveryof GPI 15427/C and GPI 16539/C, Potent Water-Soluble PARP Inhibitors,Reduces Infarct Volume Following Permanent and Transient Focal CerebralIschemia, Society for Neuroscience, Orlando Fla., October 2002; TentoriL, et al Systemic administration of the PARP-1 inhibitor GPI 15427increases the anti-tumor activity of temozolomide against metastaticmelanoma. Medical Science Monitor, volume 9, supplement 1, p 34, 2003;and Tentori et al, Poly(ADP-Ribose) Polymerase Inhibitor to IncreaseTemozolomide Efficacy Against Melanoma, Glioma and Lymphoma at the CNSSite AACR poster, April 2003, U.S. Pat. Nos. 6,348,475, 6,545,011,RE36,397, U.S. Pat. Nos. 6,380,211, 6,235,748, 6,121,278, 6,197,785,6,380,193, 6,346,536, 6,514,983, 6,306,889, 6,387,902, 6,201,020, and6,291,425, the entire contents of which patents, patent application andpublications are herein incorporated by reference, as though set forthherein in full.

Other PARP inhibitors may be available from commercial suppliers or canbe readily prepared by an ordinarily skilled artisan using standardtechniques such as those disclosed in U.S. Pat. No. 6,291,425, theentire contents of which reference are herein incorporated by referenceas though set forth herein in full.

PARP (EC 2.4.2.30), also known as PARS (for poly(ADP-ribose)synthetase), or ADPRT (for NAD:protein (ADP-ribosyl)transferase(polymerising)), or pADPRT (for poly(ADP-ribose) transferase), is amajor nuclear protein of 116 kDa. It is present in almost alleukaryotes. The enzyme synthesizes poly(ADP-ribose), a branched polymerthat can consist of over 200 ADP-ribose units from NAD. The proteinacceptors of poly(ADP-ribose) are directly or indirectly involved inmaintaining DNA integrity. They include histones, topoisomerases, DNAand RNA polymerases, DNA ligases, and Ca²⁺- and Mg²⁺-dependentendonucleases. PARP protein is expressed at a high level in manytissues, most notably in the immune system, heart, brain and germ-linecells. Under normal physiological conditions, there is minimal PARPactivity. However, DNA damage causes an immediate activation of PARP byup to 500-fold. Among the many functions attributed to PARP is its majorrole in facilitating DNA repair by ADP-ribosylation and thereforeco-ordinating a number of DNA repair proteins. As a result of PARPactivation, NAD levels significantly decline. While many endogenous andexogenous agents have been shown to damage DNA and activate PARP,peroxynitrite, formed from a combination of nitric oxide (NO) andsuperoxide, appears to be a main perpetrator responsible for variousreported disease conditions in vivo, e.g., during shock andinflammation.

Extensive PARP activation leads to severe depletion of NAD in cellssuffering from massive DNA damage. The short life of poly(ADP-ribose)(half-life <1 min) results in a rapid turnover rate. Oncepoly(ADP-ribose) is formed, it is quickly degraded by the constitutivelyactive poly(ADP-ribose) glycohydrolase (PARG), together withphosphodiesterase and (ADP-ribose) protein lyase. PARP and PARG form acycle that converts a large amount of NAD to ADP-ribose. In less than anhour, over-stimulation of PARP can cause a drop of NAD and ATP to lessthan 20% of the normal level. Such a scenario is especially detrimentalduring ischaemia when deprivation of oxygen has already drasticallycompromised cellular energy output. Subsequent free radical productionduring reperfusion is assumed to be a major cause of tissue damage. Partof the ATP drop, which is typical in many organs during ischaemia andreperfusion, could be linked to NAD depletion due to poly(ADP-ribose)turnover. Thus, PARP or PARG inhibition is expected to preserve thecellular energy level to potentiate the survival of ischaemic tissuesafter insult.

Poly(ADP-ribose) synthesis is also involved in the induced expression ofa number of genes essential for inflammatory response. PARP inhibitorssuppress production of inducible nitric oxide synthase (iNOS) inmacrophages, P-type selectin and intercellular adhesion molecule-1(ICAM-1) in endothelial cells. Such activity underlies the stronganti-inflammation effects exhibited by PARP inhibitors. PARP inhibitionis able to reduce necrosis by preventing translocation and infiltrationof neutrophils to the injured tissues. (Zhang, J. “PARP inhibition: anovel approach to treat ischaemia/reperfusion and inflammation-relatedinjuries”, Chapter 10 in Emerging Drugs (1999) 4: 209-221 AshleyPublications Ltd., and references cited therein.)

PARP production is activated by damaged DNA fragments which, onceactivated, catalyzes the attachment of up to 100 ADP-ribose units to avariety of nuclear proteins, including histones and PARP itself Duringmajor cellular stresses the extensive activation of PARP can rapidlylead to cell damage or death through depletion of energy stores. As fourmolecules of ATP are consumed for every molecule of NAD (the source ofADP-ribose and PARP substrate) regenerated, NAD is depleted by massivePARP activation and, in the efforts to re-synthesize NAD, ATP may alsobe depleted.

It has been reported that PARP activation plays a key role in both NMDA-and NO-induced neurotoxicity. This has been demonstrated in corticalcultures and in hippocampal slices wherein prevention of toxicity isdirectly correlated to PARP inhibition potency (Zhang et al., “NitricOxide Activation of Poly(ADP-Ribose) Synthetase in Neurotoxicity”,Science, 263:687-89 (1994) and Wallis et al., “Neuroprotection AgainstNitric Oxide Injury with Inhibitors of ADP-Ribosylation”, NeuroReport,5:3, 245-48 (1993)). The potential role of PARP inhibitors in treatingneurodegenerative diseases and head trauma has thus been recognized evenif the exact mechanism of action has not yet been elucidated (Endres etal., “Ischemic Brain Injury is Mediated by the Activation ofPoly(ADP-Ribose)Polymerase”, J. Cereb. Blood Flow Metabol., 17:1143-51(1997) and Wallis et al., “Traumatic Neuroprotection with Inhibitors ofNitric Oxide and ADP-Ribosylation, Brain Res., 710:169-77 (1996)).

Similarly, it has been demonstrated that single injections of PARPinhibitors have reduced the infarct size caused by ischemia andreperfusion of the heart or skeletal muscle in rabbits. In thesestudies, a single injection 3-amino-benzamide (10 mg/kg), either oneminute before occlusion or one minute before reperfusion, caused similarreductions in infarct size in the heart (32-42%) while1,5-dihydroxyisoquinoline (1 mg/kg), another PARP inhibitor, reducedinfarct size by a comparable degree (38-48%). Thiemermann et al.,“Inhibition of the Activity of Poly(ADP Ribose) Synthetase ReducesIschemia-Reperfusion Injury in the Heart and Skeletal Muscle”, Proc.Natl. Acad. Sci. USA, 94:679-83 (1997). These results make it reasonableto suspect that PARP inhibitors could salvage previously ischemic heartor skeletal muscle tissue.

PARP activation can also be used as a measure of damage followingneurotoxic insults following over-exposure to any of glutamate (via NMDAreceptor stimulation), reactive oxygen intermediates, amyloid β-protein,N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) or its activemetabolite N-methyl-4-phenylpyridine (MPP⁺), which participate inpathological conditions such as stroke, Alzheimer's disease andParkinson's disease. Zhang et al., “Poly(ADP-Ribose) SynthetaseActivation: An Early Indicator of Neurotoxic DNA Damage”, J. Neurochem.,65:3, 1411-14 (1995). Other studies have continued to explore the roleof PARP activation in cerebellar granule cells in vitro and in MPTPneurotoxicity. Cosi et al., “Poly(ADP-Ribose) Polymerase (PARP)Revisited. A New Role for an Old Enzyme: PARP Involvement inNeurodegeneration and PARP Inhibitors as Possible NeuroprotectiveAgents”, Ann. N.Y. Acad. Sci., 825:366-79 (1997); and Cosi et al.,“Poly(ADP-Ribose) Polymerase Inhibitors Protect Against MPTP-inducedDepletions of Striatal Dopamine and Cortical Noradrenaline in C57B1/6Mice”, Brain Res., 729:264-69 (1996). Excessive neural exposure toglutamate, which serves as the predominate central nervous systemneurotransmitter and acts upon the N-methyl-D-aspartate (NMDA) receptorsand other subtype receptors, most often occurs as a result of stroke orother neurodegenerative processes. Oxygen deprived neurons releaseglutamate in great quantities during ischemic brain insult such asduring a stroke or heart attack. This excess release of glutamate inturn causes over-stimulation (excitotoxicity) of N-methyl-D-aspartate(NMDA), AMPA, Kainate and MGR receptors, which open ion channels andpermit uncontrolled ion flow (e.g., Ca²⁺ and Na⁺ into the cells and K⁺out of the cells) leading to overstimulation of the neurons. Theover-stimulated neurons secrete more glutamate, creating a feedback loopor domino effect which ultimately results in cell damage or death viathe production of proteases, lipases and free radicals. Excessiveactivation of glutamate receptors has been implicated in variousneurological diseases and conditions including epilepsy, stroke,Alzheimer's disease, Parkinson's disease, Amyotrophic Lateral Sclerosis(ALS), Huntington's disease, schizophrenia, chronic pain, ischemia andneuronal loss following hypoxia, hypoglycemia, ischemia, trauma, andnervous insult. Glutamate exposure and stimulation has also beenimplicated as a basis for compulsive disorders, particularly drugdependence. Evidence includes findings in many animal species, as wellas in cerebral cortical cultures treated with glutamate or NMDA, thatglutamate receptor antagonists (i.e., compounds which block glutamatefrom binding to or activating its receptor) block neural damagefollowing vascular stroke. Dawson et al., “Protection of the Brain fromIschemia”, Cerebrovascular Disease, 319-25 (H. Hunt Batjer ed., 1997).Attempts to prevent excitotoxicity by blocking NMDA, AMPA, Kainate andMGR receptors have proven difficult because each receptor has multiplesites to which glutamate may bind and hence finding an effective mix ofantagonists or universal antagonist to prevent binding of glutamate toall of the receptor and allow testing of this theory, has beendifficult. Moreover, many of the compositions that are effective inblocking the receptors are also toxic to animals. As such, there ispresently no known effective treatment for glutamate abnormalities.

The stimulation of NMDA receptors by glutamate, for example, activatesthe enzyme neuronal nitric oxide synthase (nNOS), leading to theformation of nitric oxide (NO), which also mediates neurotoxicity. NMDAneurotoxicity may be prevented by treatment with nitric oxide synthase(NOS) inhibitors or through targeted genetic disruption of nNOS invitro. Dawson et al., “Nitric Oxide Mediates Glutamate Neurotoxicity inPrimary Cortical Cultures”, Proc. Natl. Acad. Sci. USA, 88:6368-71(1991); and Dawson et al., “Mechanisms of Nitric Oxide-mediatedNeurotoxicity in Primary Brain Cultures”, J. Neurosci., 13:6, 2651-61(1993), Dawson et al., “Resistance to Neurotoxicityin Cortical Culturesfrom Neuronal Nitric Oxide Synthase-Deficient Mice”, J. Neurosci., 16:8,2479-87 (1996), Iadecola, “Bright and Dark Sides of Nitric Oxide inIschemic Brain Injury”, Trends Neurosci., 20:3, 132-39 (1997), Huang etal., “Effects of Cerebral Ischemia in Mice Deficient in Neuronal NitricOxide Synthase”, Science, 265:1883-85 (1994), Beckman et al.,“Pathological Implications of Nitric Oxide, Superoxide and PeroxynitriteFormation”, Biochem. Soc. Trans., 21:330-34 (1993), and Szabó et al.,“DNA Strand Breakage, Activation of Poly(ADP-Ribose) Synthetase, andCellular Energy Depletion are Involved in the Cytotoxicity inMacrophages and Smooth Muscle Cells Exposed to Peroxynitrite”, Proc.Natl. Acad. Sci. USA, 93:1753-58 (1996).

It is also known that PARP inhibitors, such as 3-amino benzamide, affectDNA repair generally in response, for example, to hydrogen peroxide orgamma-radiation. Cristovao et al., “Effect of a Poly(ADP-Ribose)Polymerase Inhibitor on DNA Breakage and Cytotoxicity Induced byHydrogen Peroxide and γ-Radiation,” Terato., Carcino., and Muta.,16:219-27 (1996). Specifically, Cristovao et al. observed aPARP-dependent recovery of DNA strand breaks in leukocytes treated withhydrogen peroxide.

PARP inhibitors have been reported to be effective in radiosensitizinghypoxic tumor cells and effective in preventing tumor cells fromrecovering from potentially lethal damage of DNA after radiationtherapy, presumably by their ability to prevent DNA repair. U.S. Pat.Nos. 5,032,617; 5,215,738; and 5,041,653.

Evidence also exists that PARP inhibitors are useful for treatinginflammatory bowel disorders, such as colitis. Salzman et al., “Role ofPeroxynitrite and Poly(ADP-Ribose)Synthase Activation ExperimentalColitis,” Japanese J. Pharm., 75, Supp. I:15 (1997). Specifically,Colitis was induced in rats by intraluminal administration of the haptentrinitrobenzene sulfonic acid in 50% ethanol. Treated rats received3-aminobenzamide, a specific inhibitor of PARP activity. Inhibition ofPARP activity reduced the inflammatory response and restored themorphology and the energetic status of the distal colon. See also,Southan et al., “Spontaneous Rearrangement of Aminoalkylithioureas intoMercaptoalkylguanidines, a Novel Class of Nitric Oxide SynthaseInhibitors with Selectivity Towards the Inducible Isoform”, Br. J.Pharm., 117:619-32 (1996); and Szabó et al., “Mercaptoethylguanidine andGuanidine Inhibitors of Nitric Oxide Synthase React with Peroxynitriteand Protect Against Peroxynitrite-induced Oxidative Damage”, J. Biol.Chem., 272:9030-36 (1997).

Evidence also exists that PARP inhibitors are useful for treatingarthritis. Szabó et al., “Protective Effects of an Inhibitor ofPoly(ADP-Ribose)Synthetase in Collagen-Induced Arthritis,” Japanese J.Pharm., 75, Supp. I:102 (1997); Szabó et al., “DNA Strand Breakage,Activation of Poly(ADP-Ribose)Synthetase, and Cellular Energy Depletionare Involved in the Cytotoxicity in Macrophages and Smooth Muscle CellsExposed to Peroxynitrite,” Proc. Natl. Acad. Sci. USA, 93:1753-58 (March1996); and Bauer et al., “Modification of Growth Related EnzymaticPathways and Apparent Loss of Tumorigenicity of a ras-transformed BovineEndothelial Cell Line by Treatment with 5-Iodo-6-amino-1,2-benzopyrone(INH2BP)”, Intl. J. Oncol., 8:239-52 (1996); and Hughes et al.,“Induction of T Helper Cell Hyporesponsiveness in an Experimental Modelof Autoimmunity by Using Nonmitogenic Anti-CD3 Monoclonal Antibody”, J.Immuno., 153:3319-25 (1994).

Further, PARP inhibitors appear to be useful for treating diabetes.Heller et al., “Inactivation of the Poly(ADP-Ribose)Polymerase GeneAffects Oxygen Radical and Nitric Oxide Toxicity in Islet Cells,” J.Biol. Chem., 270:19, 11176-80 (May 1995). Heller et al. used cells frommice with inactivated PARP genes and found that these mutant cells didnot show NAD⁺ depletion after exposure to DNA-damaging radicals. Themutant cells were also found to be more resistant to the toxicity of NO.

PARP inhibitors have been shown to be useful for treating endotoxicshock or septic shock. Zingarelli et al., “Protective Effects ofNicotinamide Against Nitric Oxide-Mediated Delayed Vascular Failure inEndotoxic Shock: Potential Involvement of PolyADP Ribosyl Synthetase,”Shock, 5:258-64 (1996), suggests that inhibition of the DNA repair cycletriggered by poly(ADP ribose) synthetase has protective effects againstvascular failure in endotoxic shock. Zingarelli et al. found thatnicotinamide protects against delayed, NO-mediated vascular failure inendotoxic shock. Zingarelli et al. also found that the actions ofnicotinamide may be related to inhibition of the NO-mediated activationof the energy-consuming DNA repair cycle, triggered by poly(ADP ribose)synthetase. Cuzzocrea, “Role of Peroxynitrite and Activation ofPoly(ADP-Ribose) Synthetase in the Vascular Failure Induced byZymosan-activated Plasma,” Brit. J. Pharm., 122:493-503 (1997).

PARP inhibitors have been used to treat cancer. Suto et al.,“Dihydroisoquinolinones: The Design and Synthesis of a New Series ofPotent Inhibitors of Poly(ADP-Ribose) Polymerase”, Anticancer Drug Des.,7:107-17 (1991). In addition, Suto et al., U.S. Pat. No. 5,177,075,discusses several isoquinolines used for enhancing the lethal effects ofionizing radiation or chemotherapeutic agents on tumor cells. Weltin etal., “Effect of 6(5H)-Phenanthridinone, an Inhibitor of Poly(ADP-ribose)Polymerase, on Cultured Tumor Cells”, Oncol. Res., 6:9, 399-403 (1994),discusses the inhibition of PARP activity, reduced proliferation oftumor cells, and a marked synergistic effect when tumor cells areco-treated with an alkylating drug.

Still another use for PARP inhibitors is the treatment of peripheralnerve injuries, and the resultant pathological pain syndrome known asneuropathic pain, such as that induced by chronic constriction injury(CCI) of the common sciatic nerve and in which transsynaptic alterationof spinal cord dorsal horn characterized by hyperchromatosis ofcytoplasm and nucleoplasm (so-called “dark” neurons) occurs. Mao et al.,Pain, 72:355-366 (1997).

PARP inhibitors have also been used to extend the lifespan andproliferative capacity of cells including treatment of diseases such asskin aging, Alzheimer's disease, atherosclerosis, osteoarthritis,osteoporosis, muscular dystrophy, degenerative diseases of skeletalmuscle involving replicative senescence, age-related musculardegeneration, immune senescence, AIDS, and other immune senescencediseases; and to alter gene expression of senescent cells. WO 98/27975.

Large numbers of known PARP inhibitors have been described in Banasik etal., “Specific Inhibitors of Poly(ADP-Ribose) Synthetase andMono(ADP-Ribosyl)-Transferase”, J. Biol. Chem., 267:3, 1569-75 (1992),and in Banasik et al., “Inhibitors and Activators of ADP-RibosylationReactions”, Molec. Cell. Biochem., 138:185-97 (1994). However, effectiveuse of these PARP inhibitors, in the ways discussed above, has beenlimited by the concurrent production of unwanted side-effects (Milam etal., “Inhibitors of Poly(Adenosine Diphosphate-Ribose) Synthesis: Effecton Other Metabolic Processes”, Science, 223:589-91 (1984)).

There continues to be a need for effective and potent PARP inhibitorswhich produce minimal side-effects. The present invention providescompounds, compositions for, and methods of, inhibiting PARP activityfor treating and/or preventing cellular, tissue and/or organ damageresulting from cell damage or death due to, for example, necrosis orapoptosis. The compounds and compositions of the present invention arespecifically useful in ameliorating, treating and/or preventing neuraltissue or cell damage, including that following focal ischemia andreperfusion injury. Generally, inhibition of PARP activity spares thecell from energy loss, preventing irreversible depolarization of theneurons and, thus, provides neuroprotection. While not wishing to bebound by any mechanistic theory, the inhibition of PARP activity by useof the compounds, compositions and methods of the present invention isbelieved to protect cells, tissue and organs by protection against theill-effects of reactive free radicals and nitric oxide. The presentinvention therefore also provides methods of treating and/or preventingcells, tissue and/or organs from reactive free radical and/or nitricoxide induced damage or injury.

The present invention provides compounds which inhibit poly(ADP-ribose)polymerase (“PARP”), compositions containing these compounds and methodsfor using these PARP inhibitors to treat, prevent and/or ameliorate theeffects of the conditions described herein.

In one embodiment, the present invention provides compounds of FormulaI:

or a pharmaceutically acceptable salt, prodrug, metabolite, or hydrate;

-   -   where:    -   R1 is H, halogen, alkoxy, or lower alkyl;    -   R2 is H, halogen, alkoxy, or lower alkyl;    -   R3 is independently H, amino, hydroxy, —N—N, halogen-substituted        amino, —O-alkyl, —O-aryl, or an optionally substituted alkyl,        alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl,        heteroaryl, —COR8, where R8 is H, —OH an optionally substituted        alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl or        heteroaryl, or —OR6 or —NR6R7 where R6 and R7 are each        independently hydrogen or an optionally substituted alkyl,        alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl or        heteroaryl;    -   R4 is independently H, amino, hydroxy, —N—N, —CO—N—N,        halogen-substituted amino, —O-alkyl, —O-aryl, or an optionally        substituted alkyl, alkenyl, alkynyl, cycloalkyl,        heterocycloalkyl, aryl, heteroaryl, —COR8, where R8 is H, —OH an        optionally substituted alkyl, alkenyl, alkynyl, cycloalkyl,        heterocycloalkyl, aryl or heteroaryl, or —OR6 or —NR6R7 where R6        and R7 are each independently hydrogen or an optionally        substituted alkyl, alkenyl, alkynyl, cycloalkyl,        heterocycloalkyl, aryl or heteroaryl; and    -   R5 is independently H, amino, hydroxy, —N—N, —CO—N—N,        halogen-substituted amino, —O-alkyl, —O-aryl, or an optionally        substituted alkyl, alkenyl, alkynyl, cycloalkyl,        heterocycloalkyl, aryl, heteroaryl, —COR8, where R8 is H, —OH an        optionally substituted alkyl, alkenyl, alkynyl, cycloalkyl,        heterocycloalkyl, aryl or heteroaryl, or —OR6 or —NR6R7 where R6        and R7 are each independently hydrogen or an optionally        substituted alkyl, alkenyl, alkynyl, cycloalkyl,        heterocycloalkyl, aryl or heteroaryl.    -   The present invention also provides compounds where    -   R1 is H, F, Cl, methoxy, or methyl;    -   R2 is H, F, Cl, methoxy, or methyl;    -   R3 is independently H, amino, hydroxy, —N—N, halogen-substituted        amino, —O-alkyl, —O-aryl, or an optionally substituted alkyl,        alkeny, —COR8, where R8 is H, —OH an optionally substituted        alkyl, or alkenyl, or —OR6 or —NR6R7 where R6 and R7 are each        independently hydrogen or an optionally substituted alkyl, or        alkenyl;    -   R4 is independently H, amino, hydroxy, —N—N, —CO—N—N,        halogen-substituted amino, —O-alkyl, —O-aryl, or an optionally        substituted alkyl, alkenyl, —COR8, where R8 is H, —OH an        optionally substituted alkyl, or alkenyl, or —OR6 or —NR6R7        where R6 and R7 are each independently hydrogen or an optionally        substituted alkyl, or alkenyl; and    -   R5 is independently H, amino, hydroxy, —N—N, —CO—N—N,        halogen-substituted amino, —O-alkyl, —O-aryl, or an optionally        substituted alkyl, alkenyl —COR8, where R8 is H, —OH an        optionally substituted alkyl, or alkenyl, or —OR6 or —NR6R7        where R6 and R7 are each independently hydrogen or an optionally        substituted alkyl, or alkenyl.

The present invention also provides the following compounds

In another embodiment, the present invention provides compounds ofFormula II:

-   -   where    -   R1 is H, halogen, alkoxy, or lower alkyl;    -   R2 is H, halogen, alkoxy, or lower alkyl;    -   R3 is independently H, amino, hydroxy, —NH—NH2,        halogen-substituted amino, —O-alkyl, —O-aryl, or an optionally        substituted alkyl, alkenyl, alkynyl, cycloalkyl,        heterocycloalkyl, aryl, heteroaryl, —COR8, where R8 is H, —OH an        optionally substituted alkyl, alkenyl, alkynyl, cycloalkyl,        heterocycloalkyl, aryl or heteroaryl, or —OR6 or —NR6R7 where R6        and R7 are each independently hydrogen or an optionally        substituted alkyl, alkenyl, alkynyl, cycloalkyl,        heterocycloalkyl, aryl or heteroaryl; and    -   R4 is independently H, amino, hydroxy, —NH—NH2, —CO—NH—NH2,        halogen-substituted amino, —O-alkyl, —O-aryl, or an optionally        substituted alkyl, alkenyl, alkynyl, cycloalkyl,        heterocycloalkyl, aryl, heteroaryl, —COR8, where R8 is H, —OH an        optionally substituted alkyl, alkenyl, alkynyl, cycloalkyl,        heterocycloalkyl, aryl or heteroaryl, or —OR6 or —NR6R7 where R6        and R7 are each independently hydrogen or an optionally        substituted alkyl, alkenyl, alkynyl, cycloalkyl,        heterocycloalkyl, aryl or heteroaryl.

In another embodiment, the present invention provides compounds where

-   -   R1 is H, F, Cl, methoxy, or methyl;    -   R2 is H, F, Cl, methoxy, or methyl;    -   R3 is independently H, amino, hydroxy, —NH—NH2,        halogen-substituted amino, —O-alkyl, —O-aryl, or an optionally        substituted alkyl, alkenyl, —COR8, where R8 is H, —OH an        optionally substituted alkyl, or alkenyl, or —OR6 or —NR6R7        where R6 and R7 are each independently hydrogen or an optionally        substituted alkyl, or alkenyl; and    -   R4 is independently H, amino, hydroxy, —NH—NH2, —CO—NH—NH2,        halogen-substituted amino, —O-alkyl, —O-aryl, or an optionally        substituted alkyl, alkenyl, alkynyl, cycloalkyl,        heterocycloalkyl, aryl, heteroaryl, —COR8, where R8 is H, —OH an        optionally substituted alkyl, or alkenyl, or —OR6 or —NR6R7        where R6 and R7 are each independently hydrogen or an optionally        substituted alkyl, or alkenyl.

In another embodiment, the present invention provides the followingcompounds

The present invention also provides the following compounds of group:

Broadly, the compounds and compositions of the present invention can beused to treat or prevent cell damage or death due to necrosis orapoptosis, cerebral ischemia and reperfusion injury or neurodegenerativediseases in an animal, such as a human. The compounds and compositionsof the present invention can be used to extend the lifespan andproliferative capacity of cells and thus can be used to treat or preventdiseases associated therewith; they alter gene expression of senescentcells; and they radiosensitize hypoxic tumor cells. Preferably, thecompounds and compositions of the invention can be used to treat orprevent tissue damage resulting from cell damage or death due tonecrosis or apoptosis, and/or effect neuronal activity, either mediatedor not mediated by NMDA toxicity. The compounds of the present inventionare not limited to being useful in treating glutamate mediatedneurotoxicity and/or NO-mediated biological pathways. Further, thecompounds of the invention can be used to treat or prevent other tissuedamage related to PARP activation, as described herein.

The present invention provides compounds which inhibit the in vitroand/or in vivo polymerase activity of poly(ADP-ribose) polymerase(PARP), and compositions containing the disclosed compounds.

The present invention provides methods to inhibit, limit and/or controlthe in vitro and/or in vivo polymerase activity of poly(ADP-ribose)polymerase (PARP) in solutions, cells, tissues, organs or organ systems.In one embodiment, the present invention provides methods of limiting orinhibiting PARP activity in a mammal, such as a human, either locally orsystemically.

The present invention provides methods to treat and/or prevent diseases,syndromes and/or conditions exacerbated by or involving the increasedgeneration of PARP. These methods involve application or administrationof the compounds of the present invention to cells, tissues, organs ororgan systems of a person in need of such treatment or prevention.

In one embodiment, the present invention provides methods to treatand/or prevent cardiovascular tissue damage resulting from cardiacischemia or reperfusion injury. Reperfusion injury, for instance, occursat the termination of cardiac bypass procedures or during cardiac arrestwhen the heart, once prevented from receiving blood, begins to reperfuseand these methods involve administration of the compounds andcompositions of the present invention preferably prior to, orimmediately subsequent to reperfusion, such that reperfusion injury isprevented, treated or reduced. The present invention also providesmethods of preventing and/or treating vascular stroke, cardiovasculardisorders

In another embodiment, the present invention provides in vitro or invivo methods to extend or increase the lifespan and/or proliferationcapacity of cells and thus also methods to treat and/or prevent diseasesassociated therewith and induced or exacerbated by cellular senescenceincluding skin aging, atherosclerosis, osteoarthritis, osteoporosis,muscular dystrophy, degenerative diseases of skeletal muscle involvingreplicative senescence, age-related muscular degeneration, immunesenescence, AIDS and other immune senescence diseases, and otherdiseases associated with cellular senescence and aging, as well as toalter the gene expression of senescent cells.

In another embodiment, the present invention provides methods oftreating or preventing or ameliorating the effect of cancer and/or toradiosensitize hypoxic tumor cells to render the tumor cells moresusceptible to radiation therapy and thereby to prevent the tumor cellsfrom recovering from potentially lethal damage of DNA after radiationtherapy. A method of this embodiment is directed to specifically andpreferentially radiosensitizing tumor cells rendering the tumor cellsmore susceptible to radiation therapy than non-tumor cells.

In another embodiment the present invention provides methods ofpreventing and/or treating vascular stroke, cardiovascular disorders; totreat other conditions and/or disorders such as age-related musculardegeneration, AIDS and other immune senescence diseases, arthritis,atherosclerosis, cachexia, cancer, degenerative diseases of skeletalmuscle involving replicative senescence, diabetes, head trauma, spinalchord injury, immune senescence, inflammatory bowel disorders (such ascolitis and Crohn's disease), acute pancreatitis, mucositis, hemorrhagicshock, splanchnic artery occlusion shock, multiple organ failure (suchas involving any of the kidney, liver, renal, pulmonary, retianl,pancreatic and/or skeletal muscles systems), acute autoimmunethyroiditis, muscular dystrophy, osteoarthritis, osteoporosis, chronicand/or acute pain (such as neuropathic pain), renal failure, retinalischemia, septic shock (such as endotoxic shock), local and/or remoteentothelial cell dysfunction (such are recognized by endo-dependentrelaxant responses and up-regulation of adhesion molecules),inflammation and skin aging.

In another embodiment of the present invention, a person diagnosed withacute retinal ischemia or acute vascular stroke is immediatelyadministered parenterally, either by intermittent or continuousintravenous administration, a compound of the present invention eitheras a single dose or a series of divided doses of the compound. Afterthis initial treatment, and depending on the person's presentingneurological symptoms, the person optionally may receive the same or adifferent compound of the invention in the form of another parenteraldose. The compound of the invention can be administered by intermittentor continuous administration via implantation of a biocompatible,biodegradable polymeric matrix delivery system containing the compound,or via a subdural pump inserted to administer the compound directly tothe infarct area of the brain.

In another embodiment, the present invention provides methods to extendthe lifespan and proliferative capacity of cells, such as, for example,in using the compounds of the invention as general mediators in thegeneration of oxidants, proinflammatory mediators and/or cytokines,and/or general mediators of leukocyte infiltration, calcium ionoverload, phospholipid peroxidaion, impaired nitric oxide metabolismand/or reduced ATP production.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 Effect of Compound 43 at 40 mg/kg IV Pre and Post Transient 3VOMCAO (Total Infarct and Regional Infarct).

The compounds of the present invention can treat or prevent tissuedamage resulting from cell damage or death due to necrosis or apoptosis;can ameliorate neural or cardiovascular tissue damage, including thatfollowing focal ischemia, myocardial infarction, and reperfusion injury;can treat various diseases and conditions caused or exacerbated by PARPactivity; can extend or increase the lifespan or proliferative capacityof cells; can alter the gene expression of senescent cells; and canradiosensitize cells. Generally, inhibition of PARP activity spares thecells from energy loss, preventing, in the case of neural cells,irreversible depolarization of the neurons, and thus, providesneuroprotection. While not being bound to any one particular theory, itis thought that PARP activation may play a common role in still otherexcitotoxic mechanisms, perhaps as yet undiscovered, in addition to theproduction of free radicals and NO.

For the foregoing reasons, the present invention further relates to amethod of administering a therapeutically effective amount of theabove-identified compounds in an amount sufficient to inhibit PARPactivity, to treat or prevent tissue damage resulting from cell damageor death due to necrosis or apoptosis, to effect a neuronal activity notmediated by NMDA toxicity, to effect a neuronal activity mediated byNMDA toxicity, to treat neural tissue damage resulting from ischemia andreperfusion injury, neurological disorders and neurodegenerativediseases; to prevent or treat vascular stroke; to treat or preventcardiovascular disorders; to treat other conditions and/or disorderssuch as age-related muscular degeneration, AIDS and other immunesenescence diseases, arthritis, atherosclerosis, ataxia telangiectasia,cachexia, cancer, degenerative diseases of skeletal muscle involvingreplicative senescence, diabetes, head trauma, immune senescence,inflammatory bowel disorders (such as colitis and Crohn's disease),muscular dystrophy, osteoarthritis, osteoporosis, chronic and/or acutepain (such as neuropathic pain), renal failure, retinal ischemia, septicshock (such as endotoxic shock), and skin aging; to extend the lifespanand proliferative capacity of cells; to alter gene expression ofsenescent cells; or to radiosensitize hypoxic tumor cells. The presentinvention also relates to treating diseases and conditions in an animalwhich comprises administering to said animal a therapeutically effectiveamount of the above-identified compounds.

The present invention relates to a method of treating, preventing orinhibiting a neurological disorder in an animal, which comprisesadministering to said animal a therapeutically effective amount of theabove-identified compounds. In a another embodiment, the neurologicaldisorder is selected from the group consisting of peripheral neuropathycaused by physical injury or disease state, traumatic brain injury,physical damage to the spinal cord, stroke associated with brain damage,focal ischemia, global ischemia, reperfusion injury, demyelinatingdisease and neurological disorder relating to neurodegeneration. Anotherembodiment is when the reperfusion injury is a vascular stroke. Yetanother embodiment is when the peripheral neuropathy is caused byGuillain-Barre syndrome. Still another embodiment is when thedemyelinating disease and neurological disorder relates toneurodegeneration. Another embodiment is when the reperfusion injury isa vascular stroke. Still another preferred embodiment is when thedemyelinating disease is multiple sclerosis. Another embodiment is whenthe neurological disorder relating to neurodegeneration is selected fromthe group consisting of Alzheimer's Disease, Parkinson's Disease, andamyotrophic lateral sclerosis.

Another embodiment is a method of treating, preventing or inhibiting acardiovascular disease in an animal, such as angina pectoris, myocardialinfarction, cardiovascular ischemia, and cardiovascular tissue damagerelated to PARP activation, by administering to said animal an effectiveamount of the compounds of the present invention.

The present invention also contemplates the use of a compound thepresent invention for inhibiting PARP activity, for treating, preventingor inhibiting tissue damage resulting from cell damage or death due tonecrosis or apoptosis, for treating, preventing or inhibiting aneurological disorder in an animal.

In another embodiment, the neurological disorder is selected from thegroup consisting of peripheral neuropathy caused by physical injury ordisease state, traumatic brain injury, physical damage to the spinalcord, stroke associated with brain damage, focal ischemia, globalischemia, reperfusion injury, demyelinating disease and neurologicaldisorder relating to neurodegeneration.

Another embodiment is when the reperfusion injury is a vascular stroke.Yet another embodiment is when the peripheral neuropathy is caused byGuillain-Barre syndrome. Still another embodiment is when thedemyelinating disease is multiple sclerosis. Another embodiment is whenthe neurological disorder relating to neurodegeneration is selected fromthe group consisting of Alzheimer's Disease, Parkinson's Disease, andamyotrophic lateral sclerosis.

The present invention also contemplates the use of a compound of thepresent invention in the preparation of a medicament for the treatmentof any of the diseases and disorders in an animal described herein.

In another embodiment, the disease or disorder is a neurologicaldisorder.

In another embodiment, the neurological disorder is selected from thegroup consisting of peripheral neuropathy caused by physical injury ordisease state, traumatic brain injury, physical damage to the spinalcord, stroke associated with brain damage, focal ischemia, globalischemia, reperfusion injury, demyelinating disease and neurologicaldisorder relating to neurodegeneration. Another embodiment is when thereperfusion injury is a vascular stroke. Yet another embodiment is whenthe peripheral neuropathy is caused by Guillain-Barre syndrome.

Still another embodiment is when the demyelinating disease is multiplesclerosis. Another embodiment is when the neurological disorder relatingto neurodegeneration is selected from the group consisting ofAlzheimer's Disease, Parkinson's Disease, and amyotrophic lateralsclerosis.

Further still, the methods of the invention can be used to treat cancerand to radiosensitize tumor cells. The term “cancer” is interpretedbroadly. The compounds of the present invention can be “anti-canceragents”, which term also encompasses “anti-tumor cell growth agents” and“anti-neoplastic agents”. For example, the methods of the invention areuseful for treating cancers and radiosensitizing tumor cells in cancerssuch as ACTH-producing tumors, acute lymphocytic leukemia, acutenonlymphocytic leukemia, cancer of the adrenal cortex, bladder cancer,brain cancer, breast cancer, cervical cancer, chronic lymphocyticleukemia, chronic myelocytic leukemia, colorectal cancer, cutaneousT-cell lymphoma, endometrial cancer, esophageal cancer, Ewing's sarcoma,gallbladder cancer, hairy cell leukemia, head & neck cancer, Hodgkin'slymphoma, Kaposi's sarcoma, kidney cancer, liver cancer, lung cancer(small and/or non-small cell), malignant peritoneal effusion, malignantpleural effusion, melanoma, mesothelioma, multiple myeloma,neuroblastoma, non-Hodgkin's lymphoma, osteosarcoma, ovarian cancer,ovary (germ cell) cancer, prostate cancer, pancreatic cancer, penilecancer, retinoblastoma, skin cancer, soft-tissue sarcoma, squamous cellcarcinomas, stomach cancer, testicular cancer, thyroid cancer,trophoblastic neoplasms, uterine cancer, vaginal cancer, cancer of thevulva and Wilm's tumor.

The methods of the present invention can also treat cancer in a mammalwith an effective amount of temozolimide and a compound of the presentinvention. The cancer can be melanoma, lymphoma, and glioblastomamultiforme.

Radiosensitizers are known to increase the sensitivity of cancerouscells to the toxic effects of electromagnetic radiation. Severalmechanisms for the mode of action of radiosensitizers have beensuggested in the literature including: hypoxic cell radiosensitizers(e.g., 2-nitroimidazole compounds, and benzotriazine dioxide compounds)promote the reoxygenation of hypoxic tissue and/or catalyze thegeneration of damaging oxygen radicals; non-hypoxic cellradiosensitizers (e.g., halogenated pyrimidines) can be analogs of DNAbases and preferentially incorporate into the DNA of cancer cells andthereby promote the radiation-induced breaking of DNA molecules and/orprevent the normal DNA repair mechanisms; and various other potentialmechanisms of action have been hypothesized for radiosensitizers in thetreatment of disease.

Many cancer treatment protocols currently employ radiosensitizersactivated by the electromagnetic radiation of x-rays. Examples of x-rayactivated radiosensitizers include, but are not limited to, thefollowing: metronidazole, misonidazole, desmethylmisonidazole,pimonidazole, etanidazole, nimorazole, mitomycin C, RSU 1069, SR 4233,EO9, RB 6145, nicotinamide, 5-bromodeoxyuridine (BUdR),5-iododeoxyuridine (IUdR), bromodeoxycytidine, fluorodeoxyuridine(FudR), hydroxyurea, cisplatin, and therapeutically effective analogsand derivatives of the same.

Photodynamic therapy (PDT) of cancers employs visible light as theradiation activator of the sensitizing agent. Examples of photodynamicradiosensitizers include the following, but are not limited to:hematoporphyrin derivatives, Photofrin, benzoporphyrin derivatives,NPe6, tin etioporphyrin SnET2, pheoborbide-a, bacteriochlorophyll-a,naphthalocyanines, phthalocyanines, zinc phthalocyanine, andtherapeutically effective analogs and derivatives of the same.

Radiosensitizers may be administered in conjunction with atherapeutically effective amount of one or more other compounds,including but not limited to: compounds which promote the incorporationof radiosensitizers to the target cells; compounds which control theflow of therapeutics, nutrients, and/or oxygen to the target cells;chemotherapeutic agents which act on the tumor with or withoutadditional radiation; or other therapeutically effective compounds fortreating cancer or other disease. Examples of additional therapeuticagents that may be used in conjunction with radiosensitizers include,but are not limited to: 5-fluorouracil, leucovorin,5′-amino-5′deoxythymidine, oxygen, carbogen, red cell transfusions,perfluorocarbons (e.g., Fluosol-DA), 2,3-DPG, BW12C, calcium channelblockers, pentoxyfylline, antiangiogenesis compounds, hydralazine, andL-BSO. Examples of chemotherapeutic agents that may be used inconjunction with radiosensitizers include, but are not limited to:adriamycin, camptothecin, carboplatin, cisplatin, daunorubicin,docetaxel, doxorubicin, interferon (alpha, beta, gamma), interleukin 2,irinotecan, paclitaxel, topotecan, and therapeutically effective analogsand derivatives of the same.

The present invention also relates to a pharmaceutical compositioncomprising (i) a therapeutically effective amount of a compound of thepresent invention and (ii) a pharmaceutically acceptable carrier.

The above discussion relating to the preferred embodiments' utility andadministration of the compounds of the present invention also applies tothe pharmaceutical composition of the present invention.

The term “pharmaceutically acceptable carrier” as used herein refers toany carrier, diluent, excipient, suspending agent, lubricating agent,adjuvant, vehicle, delivery system, emulsifier, disintegrant, absorbent,preservative, surfactant, colorant, flavorant, or sweetener.

For these purposes, the composition of the invention may be administeredorally, parenterally, by inhalation spray, adsorption, absorption,topically, rectally, nasally, bucally, vaginally, intraventricularly,via an implanted reservoir in dosage formulations containingconventional non-toxic pharmaceutically-acceptable carriers, or by anyother convenient dosage form. The term parenteral as used hereinincludes subcutaneous, intravenous, intramuscular, intraperitoneal,intrathecal, intraventricular, intrasternal, and intracranial injectionor infusion techniques.

When administered parenterally, the composition will normally be in aunit dosage, sterile injectable form (solution, suspension or emulsion)which is preferably isotonic with the blood of the recipient with apharmaceutically acceptable carrier. Examples of such sterile injectableforms are sterile injectable aqueous or oleaginous suspensions. Thesesuspensions may be formulated according to techniques known in the artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable forms may also be sterile injectable solutions orsuspensions in non-toxic parenterally-acceptable diluents or solvents,for example, as solutions in 1,3-butanediol. Among the acceptablevehicles and solvents that may be employed are water, saline, Ringer'ssolution, dextrose solution, isotonic sodium chloride solution, andHanks' solution. In addition, sterile, fixed oils are conventionallyemployed as solvents or suspending mediums. For this purpose, any blandfixed oil may be employed including synthetic mono- or di-glycerides,corn, cottonseed, peanut, and sesame oil. Fatty acids such as ethyloleate, isopropyl myristate, and oleic acid and its glyceridederivatives, including olive oil and castor oil, especially in theirpolyoxyethylated versions, are useful in the preparation of injectables.These oil solutions or suspensions may also contain long-chain alcoholdiluents or dispersants.

Sterile saline is a preferred carrier, and the compounds are oftensufficiently water soluble to be made up as a solution for allforeseeable needs. The carrier may contain minor amounts of additives,such as substances that enhance solubility, isotonicity, and chemicalstability, e.g., anti-oxidants, buffers and preservatives.

Formulations suitable for nasal or buccal administration (such asself-propelling powder dispensing formulations) may comprise about 0.1%to about 5% w/w, for example 1% w/w of active ingredient. Theformulations for human medical use of the present invention comprise anactive ingredient in association with a pharmaceutically acceptablecarrier therefore and optionally other therapeutic ingredient(s).

When administered orally, the composition will usually be formulatedinto unit dosage forms such as tablets, cachets, powder, granules,beads, chewable lozenges, capsules, liquids, aqueous suspensions orsolutions, or similar dosage forms, using conventional equipment andtechniques known in the art. Such formulations typically include asolid, semisolid, or liquid carrier. Exemplary carriers include lactose,dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calciumphosphate, mineral oil, cocoa butter, oil of theobroma, alginates,tragacanth, gelatin, syrup, methyl cellulose, polyoxyethylene sorbitanmonolaurate, methyl hydroxybenzoate, propyl hydroxybenzoate, talc,magnesium stearate, and the like.

The composition of the invention can be administered as a capsule ortablet containing a single or divided dose of the inhibitor. Thecomposition can also be administered as a sterile solution, suspension,or emulsion, in a single or divided dose. Tablets may contain carrierssuch as lactose and corn starch, and/or lubricating agents such asmagnesium stearate. Capsules may contain diluents including lactose anddried corn starch.

A tablet may be made by compressing or molding the active ingredientoptionally with one or more accessory ingredients. Compressed tabletsmay be prepared by compressing, in a suitable machine, the activeingredient in a free-flowing form such as a powder or granules,optionally mixed with a binder, lubricant, inert diluent, surfaceactive, or dispersing agent. Molded tablets may be made by molding in asuitable machine, a mixture of the powdered active ingredient and asuitable carrier moistened with an inert liquid diluent.

The compounds of this invention may also be administered rectally in theform of suppositories. These compositions can be prepared by mixing thedrug with a suitable non-irritating excipient which is solid at roomtemperature, but liquid at rectal temperature, and, therefore, will meltin the rectum to release the drug. Such materials include cocoa butter,beeswax, and polyethylene glycols.

Compositions and methods of the invention also may utilize controlledrelease technology. Thus, for example, the inventive compounds may beincorporated into a hydrophobic polymer matrix for controlled releaseover a period of days. The composition of the invention may then bemolded into a solid implant, or externally applied patch, suitable forproviding efficacious concentrations of the PARP inhibitors over aprolonged period of time without the need for frequent re-dosing. Suchcontrolled release films are well known to the art. Other embodimentsare transdermal delivery systems. Other examples of polymers commonlyemployed for this purpose that may be used in the present inventioninclude nondegradable ethylene-vinyl acetate copolymer an degradablelactic acid-glycolic acid copolymers which may be used externally orinternally. Certain hydrogels such as poly(hydroxyethylmethacrylate) orpoly(vinylalcohol) also may be useful, but for shorter release cyclesthan the other polymer release systems, such as those mentioned above.

In another embodiment, the carrier is a solid biodegradable polymer ormixture of biodegradable polymers with appropriate time releasecharacteristics and release kinetics. The composition of the inventionmay then be molded into a solid implant suitable for providingefficacious concentrations of the compounds of the invention over aprolonged period of time without the need for frequent re-dosing. Thecomposition of the present invention can be incorporated into thebiodegradable polymer or polymer mixture in any suitable manner known toone of ordinary skill in the art and may form a homogeneous matrix withthe biodegradable polymer, or may be encapsulated in some way within thepolymer, or may be molded into a solid implant.

In one embodiment, the biodegradable polymer or polymer mixture is usedto form a soft “depot” containing the pharmaceutical composition of thepresent invention that can be administered as a flowable liquid, forexample, by injection, but which remains sufficiently viscous tomaintain the pharmaceutical composition within the localized area aroundthe injection site. The degradation time of the depot so formed can bevaried from several days to a few years, depending upon the polymerselected and its molecular weight. By using a polymer composition ininjectable form, even the need to make an incision may be eliminated. Inany event, a flexible or flowable delivery “depot” will adjust to theshape of the space it occupies with the body with a minimum of trauma tosurrounding tissues. The pharmaceutical composition of the presentinvention is used in amounts that are therapeutically effective, and maydepend upon the desired release profile, the concentration of thepharmaceutical composition required for the sensitizing effect, and thelength of time that the pharmaceutical composition has to be releasedfor treatment.

The PARP inhibitors are used in the composition in amounts that aretherapeutically effective. The compositions may be sterilized and/orcontain adjuvants, such as preserving, stabilizing, welling, oremulsifying agents, solution promoters, salts for regulating the osmoticpressure, and/or buffers. In addition, they may also contain othertherapeutically valuable substances. The compositions are preparedaccording to conventional mixing, granulating, or coating methods, andcontain about 0.1 to 75% by weight, preferably about 1 to 50% by weight,of the active ingredient.

To be effective therapeutically as central nervous system targets, thecompounds of the present invention should readily penetrate theblood-brain barrier when peripherally administered. Compounds whichcannot penetrate the blood-brain barrier can be effectively administeredby an intraventricular route or other appropriate delivery systemsuitable for administration to the brain.

Doses of the compounds include pharmaceutical dosage units comprising anefficacious quantity of active compound. By an efficacious quantity ismeant a quantity sufficient to inhibit PARP and derive its beneficialeffects through administration of one or more of the pharmaceuticaldosage units. Also, the dose is sufficient to prevent or reduce theeffects of vascular stroke or other neurodegenerative diseases.

For medical use, the amount required of the active ingredient to achievea therapeutic effect will vary with the particular compound, the routeof administration, the mammal under treatment, and the particulardisorder or disease being treated. A suitable systematic dose of acompound of the present invention or a pharmacologically acceptable saltthereof for a mammal suffering from, or likely to suffer from, any ofcondition as described hereinbefore is in the range of about 0.1 mg/kgto about 100 mg/kg of the active ingredient compound, the dosage beingabout 1 to about 10 mg/kg.

It is understood, however, that a specific dose level for any particularpatient will depend upon a variety of factors including the activity ofthe specific compound employed, the age, body weight, general health,sex, diet, time of administration, rate of excretion, drug combination,and the severity of the particular disease being treated and form ofadministration.

It is understood that the ordinarily skilled physician or veterinarianwill readily determine and prescribe the effective amount of thecompound for prophylactic or therapeutic treatment of the condition forwhich treatment is administered. In so proceeding, the physician orveterinarian could employ an intravenous bolus followed by anintravenous infusion and repeated administrations, parenterally ororally, as considered appropriate. While it is possible for an activeingredient to be administered alone, it is preferable to present it as aformulation.

When preparing dosage forms incorporating the compositions of theinvention, the compounds may also be blended with conventionalexcipients such as binders, including gelatin, pregelatinized starch,and the like; lubricants, such as hydrogenated vegetable oil, stearicacid, and the like; diluents, such as lactose, mannose, and sucrose;disintegrants, such as carboxymethylcellulose and sodium starchglycolate; suspending agents, such as povidone, polyvinyl alcohol, andthe like; absorbants, such as silicon dioxide; preservatives, such asmethylparaben, propylparaben, and sodium benzoate; surfactants, such assodium lauryl sulfate, polysorbate 80, and the like; colorants such asF. D. & C. dyes and lakes; flavorants; and sweeteners.

The present invention relates to the use of compounds of the presentinvention in the preparation of a medicament for the treatment of anydisease or disorder in an animal described herein.

As used herein, “alkyl” means a branched or unbranched saturatedhydrocarbon chain comprising a designated number of carbon atoms. Forexample, C₁-C₆ straight or branched alkyl hydrocarbon chain contains 1to 6 carbon atoms, and includes but is not limited to substituents suchas methyl, ethyl, propyl, iso-propyl, butyl, iso-butyl, tert-butyl,n-pentyl, n-hexyl, and the like, unless otherwise indicated.

“Alkenyl” means a branched or unbranched unsaturated hydrocarbon chaincomprising a designated number of carbon atoms. For example, C₂-C₆straight or branched alkenyl hydrocarbon chain contains 2 to 6 carbonatoms having at least one double bond, and includes but is not limitedto substituents such as ethenyl, propenyl, isopropenyl, butenyl,iso-butenyl, tert-butenyl, n-pentenyl, n-hexenyl, and the like, unlessotherwise indicated.

“Alkoxy”, means the group —OR wherein R is alkyl as herein defined. Rcan also be a branched or unbranched saturated hydrocarbon chaincontaining 1 to 6 carbon atoms.

“Cyclo”, used herein as a prefix, refers to a structure characterized bya closed ring.

“Halo” means at least one fluoro, chloro, bromo, or iodo moiety, unlessotherwise indicated.

“Amino” compounds include amine (NH₂) as well as substituted amino.

“Ar”, “aryl” or “heteroaryl” means a moiety which is substituted orunsubstituted, especially a cyclic or fused cyclic ring and includes amono-, bi-, or tricyclic, carbo- or heterocyclic ring, wherein the ringis either unsubstituted or substituted in one to five position(s) withhalo, haloalkyl, hydroxyl, nitro, trifluoromethyl, C₁-C₆ straight orbranched chain alkyl, C₂-C₆ straight or branched chain alkenyl, C₁-C₆alkoxy, C₂-C₆ alkenyloxy, phenoxy, benzyloxy, amino, thiocarbonyl,ester, thioester, cyano, imino, alkylamino, aminoalkyl, sulfhydryl,thioalkyl, and sulfonyl; wherein the individual ring sizes are 5-8members; wherein the heterocyclic ring contains 1-4 heteroatom(s)selected from the group consisting of O, N, or S; wherein aromatic ortertiary alkyl amines are optionally oxidized to a correspondingN-oxide. Heteroaryls may be attached to other rings or substitutedthrough the heteroatom and/or carbon atom of the ring. Aryl orheteroaryl moieties include but are not limited to phenyl, benzyl,naphthyl, pyrrolyl, pyrrolidinyl, pyridinyl, pyrimidinyl, purinyl,quinolinyl, isoquinolinyl, furyl, thiophenyl, imidazolyl, oxazolyl,thiazolyl, pyrazolyl, and thienyl.

“Phenyl” includes all possible isomeric phenyl radicals, optionallymonosubstituted or multi-substituted with substituents selected from thegroup consisting of amino, trifluoromethyl, C₁-C₆ straight or branchedchain alkyl, C₂-C₆ straight or branched chain alkenyl, carbonyl,thiocarbonyl, ester, thioester, alkoxy, alkenoxy, cyano, nitro, imino,alkylamino, aminoalkyl, sulfhydryl, thioalkyl, sulfonyl, hydroxy, halo,haloalkyl, NR₂ wherein R₂ is selected from the group consisting ofhydrogen, (C₁-C₆)-straight or branched chain alkyl, (C₃-C₆) straight orbranched chain alkenyl or alkynyl, and (C₁-C₄) bridging alkyl whereinsaid bridging alkyl forms a heterocyclic ring starting with the nitrogenof NR₁ and ending with one of the carbon atoms of said alkyl or alkenylchain, and wherein said heterocyclic ring is optionally fused to an Argroup.

Cycloalkyl optionally containing at least one heteroatom includessaturated C₃-C₈ rings, such as C₅ or C₆ rings, wherein at 1-4heteroatoms selected from O, N or S may be optionally substituted for acarbon atom of the ring. Cycloalkyls optionally containing at least oneheteroatom, as described above, may be substituted by or fused to atleast one 5 or 6 membered aryl or heteroaryl. Other cycloalkylscontaining a heteroatom include pyrrolidinyl, imidazolidinyl,pyrazolidinyl, piperidinyl, piperazinyl, morpholino and thiomorpholino.

The compounds of the present invention possess one or more asymmetriccenter(s) and thus can be produced as mixtures (racemic and non-racemic)of stereoisomers, or as individual enantiomers or diastereomers. Theindividual stereoisomers may be obtained by using an optically activestarting material, by resolving a racemic or non-racemic mixture of anintermediate at some appropriate stage of the synthesis, or byresolution of the compound of the present invention. It is understoodthat the individual stereoisomers as well as mixtures (racemic andnon-racemic) of stereoisomers are encompassed by the scope of thepresent invention.

“Isomers” are different compounds that have the same molecular formulaand includes cyclic isomers such as (iso)indole and other isomeric formsof cyclic moieties. “Stereoisomers” are isomers that differ only in theway the atoms are arranged in space. “Enantiomers” are a pair ofstereoisomers that are non-superimposable mirror images of each other.“Diastereoisomers” are stereoisomers which are not mirror images of eachother. “Racemic mixture” means a mixture containing equal parts ofindividual enantiomers. “Non-racemic mixture” is a mixture containingunequal parts of individual enantiomers or stereoisomers.

The compounds of the invention are useful in a free base form, in theform of pharmaceutically acceptable salts, pharmaceutically acceptablehydrates, pharmaceutically acceptable esters, pharmaceuticallyacceptable solvates, pharmaceutically acceptable prodrugs,pharmaceutically acceptable metabolites, and in the form ofpharmaceutically acceptable stereoisomers. These forms are all withinthe scope of the invention. In practice, the use of these forms amountsto use of the neutral compound.

“Pharmaceutically acceptable salt”, “hydrate”, “ester” or “solvate”refers to a salt, hydrate, ester, or solvate of the inventive compoundswhich possesses the desired pharmacological activity and which isneither biologically nor otherwise undesirable. Organic acids can beused to produce salts, hydrates, esters, or solvates such as acetate,adipate, alginate, aspartate, benzoate, benzenesulfonate,p-toluenesulfonate, bisulfate, sulfamate, sulfate, naphthylate,butyrate, citrate, camphorate, camphorsulfonate,cyclopentane-propionate, digluconate, dodecylsulfate, ethanesulfonate,fumarate, glucoheptanoate, glycerophosphate, hemisulfate heptanoate,hexanoate, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate,2-naphthalenesulfonate, nicotinate, oxalate, tosylate and undecanoate.Inorganic acids can be used to produce salts, hydrates, esters, orsolvates such as hydrochloride, hydrobromide, hydroiodide, andthiocyanate.

Examples of suitable base salts, hydrates, esters, or solvates includehydroxides, carbonates, and bicarbonates of ammonia, alkali metal saltssuch as sodium, lithium and potassium salts, alkaline earth metal saltssuch as calcium and magnesium salts, aluminum salts, and zinc salts.

Salts, hydrates, esters, or solvates may also be formed with organicbases. Organic bases suitable for the formation of pharmaceuticallyacceptable base addition salts, hydrates, esters, or solvates of thecompounds of the present invention include those that are non-toxic andstrong enough to form such salts, hydrates, esters, or solvates. Forpurposes of illustration, the class of such organic bases may includemono-, di-, and trialkylamines, such as methylamine, dimethylamine,triethylamine and dicyclohexylamine; mono-, di- ortrihydroxyalkylamines, such as mono-, di-, and triethanolamine; aminoacids, such as arginine and lysine; guanidine; N-methyl-glucosamine;N-methyl-glucamine; L-glutamine; N-methyl-piperazine; morpholine;ethylenediamine; N-benzyl-phenethylamine;(trihydroxy-methyl)aminoethane; and the like. See, for example,“Pharmaceutical Salts,” J. Pharm. Sci., 66:1, 1-19 (1977). Accordingly,basic nitrogen-containing groups can be quaternized with agentsincluding: lower alkyl halides such as methyl, ethyl, propyl, and butylchlorides, bromides and iodides; dialkyl sulfates such as dimethyl,diethyl, dibutyl and diamyl sulfates; long chain halides such as decyl,lauryl, myristyl and stearyl chlorides, bromides and iodides; andaralkyl halides such as benzyl and phenethyl bromides.

The acid addition salts, hydrates, esters, or solvates of the basiccompounds may be prepared either by dissolving the free base of a PARPinhibitor of the present invention in an aqueous or an aqueous alcoholsolution or other suitable solvent containing the appropriate acid orbase, and isolating the salt by evaporating the solution. Alternatively,the free base of the PARP inhibitor of the present invention can bereacted with an acid, as well as reacting the PARP inhibitor having anacid group thereon with a base, such that the reactions are in anorganic solvent, in which case the salt separates directly or can beobtained by concentrating the solution.

“Pharmaceutically acceptable prodrug” refers to a derivative of theinventive compounds which undergoes biotransformation prior toexhibiting its pharmacological effect(s). The prodrug is formulated withthe objective(s) of improved chemical stability, improved patientacceptance and compliance, improved bioavailability, prolonged durationof action, improved organ selectivity, improved formulation (e.g.,increased hydrosolubility), and/or decreased side effects (e.g.,toxicity). The prodrug can be readily prepared from the inventivecompounds using methods known in the art, such as those described byBurger's Medicinal Chemistry and Drug Chemistry, Fifth Ed., Vol. 1, pp.172-178, 949-982 (1995). For example, the inventive compounds can betransformed into prodrugs by converting one or more of the hydroxy orcarboxy groups into esters.

“Pharmaceutically acceptable metabolite” refers to drugs that haveundergone a metabolic transformation. After entry into the body, mostdrugs are substrates for chemical reactions that may change theirphysical properties and biologic effects. These metabolic conversions,which usually affect the polarity of the compound, alter the way inwhich drugs are distributed in and excreted from the body. However, insome cases, metabolism of a drug is required for therapeutic effect. Forexample, anticancer drugs of the antimetabolite class must be convertedto their active forms after they have been transported into a cancercell. Since most drugs undergo metabolic transformation of some kind,the biochemical reactions that play a role in drug metabolism may benumerous and diverse. The main site of drug metabolism is the liver,although other tissues may also participate.

The term “neurodegenerative diseases” includes, but is not limited toAlzheimer's disease, Parkinson's disease and Huntington's disease.

The term “nervous insult” refers to any damage to nervous tissue and anydisability or death resulting therefrom. The cause of nervous insult maybe metabolic, toxic, neurotoxic, iatrogenic, thermal or chemical, andincludes without limitation, ischemia, hypoxia, cerebrovascularaccident, trauma, surgery, pressure, mass effect, hemmorrhage,radiation, vasospasm, neurodegenerative disease, infection, Parkinson'sdisease, amyotrophic lateral sclerosis (ALS), myelination/demyelinationprocess, epilepsy, cognitive disorder, glutamate abnormality andsecondary effects thereof.

The term “neuroprotective” refers to the effect of reducing, arrestingor ameliorating nervous insult, and protecting, resuscitating, orreviving nervous tissue that has suffered nervous insult.

The term “preventing neurodegeneration” includes the ability to preventa neurodegenerative disease or preventing further neurodegeneration inpatients who are already suffering from or have symptoms of aneurodegenerative disease.

The term “treating” refers to:

-   -   (i) preventing a disease, disorder or condition from occurring        in an animal that may be predisposed to the disease, disorder        and/or condition, but has not yet been diagnosed as having it;    -   (ii) inhibiting the disease, disorder or condition, i.e.,        arresting its development; and    -   (iii) relieving the disease, disorder or condition, i.e.,        causing regression of the disease, disorder and/or condition.

The term “neural tissue damage resulting from ischemia and reperfusioninjury and neurodegenerative diseases” includes damage due toneurotoxicity, such as seen in vascular stroke and global and focalischemia.

The term “ischemia” relates to localized tissue anemia due toobstruction of the inflow of arterial blood. Global ischemia occursunder conditions in which blood flow to the entire brain ceases for aperiod of time, such as may result from cardiac arrest. Focal ischemiaoccurs under conditions in which a portion of the brain is deprived ofits normal blood supply, such as may result from thromboembolyticocclusion of a cerebral vessel, traumatic head injury, edema, and braintumors.

The term “cardiovascular disease” relates to myocardial infarction,angina pectoris, vascular or myocardial ischemia, and related conditionsas would be known by those of skill in the art which involve dysfunctionof or tissue damage to the heart or vasculature, and especially, but notlimited to, tissue damage related to PARP activation.

The term “radiosensitizer”, as used herein, is defined as a molecule,such as a low molecular weight molecule, administered to animals intherapeutically effective amounts to increase the sensitivity of thecells to be radiosensitized to electromagnetic radiation and/or topromote the treatment of diseases which are treatable withelectromagnetic radiation. Diseases which are treatable withelectromagnetic radiation include neoplastic diseases, benign andmalignant tumors, and cancerous cells. Electromagnetic radiationtreatment of other diseases not listed herein are also contemplated bythe present invention. The terms “electromagnetic radiation” and“radiation” as used herein includes, but is not limited to, radiationhaving the wavelength of 10⁻²⁰ to 10⁰ meters. Preferred embodiments ofthe present invention employ the electromagnetic radiation of:gamma-radiation (10⁻²⁰ to 10⁻¹³ m) x-ray radiation (10⁻¹¹ to 10⁻⁹ m),ultraviolet light (10 nm to 400 nm), visible light (400 nm to 700 nm),infrared radiation (700 nm to 1.0 mm), or microwave radiation (1 mm to30 cm).

Many of the PARP inhibitors can be synthesized by known methods fromstarting materials that are known, may be available commercially, or maybe prepared by methods used to prepare corresponding compounds in theliterature. See, for example, Suto et al., “Dihydroiso-quinolinones: TheDesign and Synthesis of a New Series of Potent Inhibitors ofPoly(ADP-ribose) Polymerase”, Anticancer Drug Des., 6:107-17 (1991),which discloses processes for synthesizing a number of different PARPinhibitors.

The quinazoline-one and phthalazin-one derivatives of this invention arerepresented by previously defined formulas I and II. As an example, thederivatives of this invention can be prepared in a conventional manneras illustrated below by Schemes 1-8. The rings on the quinazoline-oneand phthalazin-one derivatives may be generically substituted as setforth in formula I and II. Such starting derivatives are known in thechemistry literature and accessible by processes known to one skilled inthe art.

The diazabenzo[de]anthracen-3-one derivatives of this invention can beprepared in a conventional manner as illustrated below by Schemes 9-11.

Other manners, variations or sequences of preparing the compounds of thepresent invention will be readily apparent to those skilled in the art.

The compounds of the present invention may be useful in the free baseform, in the form of base salts where possible, and in the form ofaddition salts, as well as in the free acid form. All these forms arewithin the scope of this invention. In practice, use of the salt formamounts to use of the base form. Pharmaceutically acceptable saltswithin the scope of this invention are those derived from mineral acidssuch as hydrochloric acid and sulfuric acid; and organic acids such asethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, andthe like, giving the hydrochloride, sulfonate, ethanesulfonate,benzenesulfonate, p-toluenesulfonate, and the like respectively, orthose derived from bases such as suitable organic and inorganic bases.Examples of pharmaceutically acceptable base addition salts withcompounds of the present invention include organic bases which arenontoxic and strong enough to form such salts. These organic bases andthe use thereof are readily understood by those skilled in the art.Merely for the purpose of illustration, such organic bases may includemono-, di-, and trialkylamines, such as methylamine, diethylamine andtriethylamine; mono-, di-, or trihydroxyalkylamines such as mono-, di-,and triethanolamine; amino acids such as arginine, and lysine;guanidine; N-methylglucosamine; N-methylgiucamine; L-glutamine;N-methylpiperazine; morpholine; ethylenedianane; N-benzylphenethylamine;tris(hydroxymethyl)antinoethane; and the like.

The acid addition salts of the basic compounds may be prepared bydissolving the free base of the compound of the present invention inaqueous or aqueous alcohol solution or other suitable solventscontaining the appropriate acid or base and isolating the salt byevaporating the solution, or by reacting the free base of the compoundof the present invention with an acid as well as reacting the compoundof the present invention having an acid group thereon with a base suchthat the reactions are in an organic solvent, in which case the saltseparates directly or can be obtained by concentration of the solution.

The compounds of this invention contain one or more asymmetric carbonatoms. Therefore, the invention includes the individual stereoisomersand mixtures thereof as well as the racemic compounds. The individualisomers may be prepared or isolated by methods known in the art.

The compounds of the invention exhibit pharmacological activity and are,therefore, useful as pharmaceuticals. Additionally, the compoundsexhibit central nervous and cardiac vesicular system activity.

PARP Assays

IC₅₀

A convenient method to determine IC₅₀ of a PARP inhibitor compound is aPARP assay using purified recombinant human PARP from Trevigan(Gaithersburg, Md.), as follows: The PARP enzyme assay is set up on icein a volume of 100 microliters consisting of 100 mM Tris-HCl (pH 8.0), 1mM MgCl₂, 28 mM KCl, 28 mM NaCl, 0.1 mg/ml of DNase I activated herringsperm DNA (Sigma, Mo.), 3.0 micromolar [3H]nicotinamide adeninedinucleotide (470 mci/mmole), 7 micrograms/ml PARP enzyme, and variousconcentrations of the compounds to be tested. The reaction is initiatedby incubating the mixture at 25° C. After 15 minutes of incubation, thereaction is terminated by adding 500 microliters of ice cold 20% (w/v)trichloroacetic acid. The precipitate formed is transferred onto a glassfiber filter (Packard Unifilter-GF/B) and washed three times withethanol. After the filter is dried, the radioactivity is determined byscintillation counting. The compounds of this invention were found tohave potent enzymatic activity in the range of a few nM to 20 μM in IC₅₀in this inhibition assay.

EC₅₀

We used a H₂O₂ induced cell death assay to determine the cytoprotectiveeffect of PARP inhibitors. P388D1 cells (CCL-46, ATCC), derived from amurine macrophage-like tumor, were maintained in Dulbeco's ModifiedEagle Medium (DMEM) with 10% horse serum and 2 mM L-glutamine. Thecytotoxicity assay was set up in a 96-well plate. In each well, 190 ulcells were seeded at 2×10⁶/ml density. To determine the EC₅₀, theconcentration of a compound required to achieve 50% reduction of celldeath, a dose response experiment was conducted. PARP inhibitors wereadded to the media to a final concentration of 0.01, 0.03, 0.1, 0.3, 1,3, 10, 30 uM. After 15 min incubation with a PARP inhibitor, 5 ul offreshly prepared H₂O₂ were added to the cells to a final concentrationof 2 mM. Cells were returned to 37° C. incubator for 4 h. At the end ofincubation, 25 ul of supernatant were sampled from the cell media todetermine the level of lactate dehydrogenase (LDH) released from deadcells. The LDH activity was determined by monitoring the rate ofdecrease of NADH absorbency at 340 nm. The group without drug treatmentwas used to calculate total cell death due to H₂O₂ treatment. Each datapoint was an average of quadruplicate. The EC₅₀ was determined from adose response curve.

Using the PARP assays described above, approximate IC₅₀ and EC₅₀ valueswere obtained for the following compounds:

TABLE I Structure IC50 (μM) EC50 (μM)

20

20

0.749

0.039 0.35

0.014 0.1

2.79

0.026 0.51

0.16 1.7

0.091 0.64

0.021 0.24

0.12

0.026 0.36

0.087 0.01

0.019 0.13

0.019 0.12

0.018

20

0.018

TABLE II Structure IC50 (μM) EC50 (μM)

0.1 0.17

0.035 2.08

0.021 5.27

0.019 2.08

0.067 0.3

0.03 0.08

0.035 1

0.074 0.13

0.06 0.12

0.101 0.08

0.128 0.075

0.165 0.47

0.092 0.41

0.038

0.413

0.125

0.086 0.52

0.177

0.167

0.275

0.034 0.75

1.3

0.244

0.089

0.036 0.3

0.048 0.27

EXAMPLE 1 Preparation of Quinazoline Derivatives (5a, 5b, 5c, and 6)(Scheme 1)

Dimethyl 3-aminophthalate (1) To a solution of dimethyl 3-nitrophthalate(12 g, 50 mmol) in EtOAc (200 mL) was added 10% Pd/C (3 g). Theresulting mixture was hydrogenated under H₂ (50 psi) on a Parrhydrogenation apparatus at room temperature overnight. The catalyst wasfiltered off on a pad of celite and the filtrate was concentrated invacuum to afford a yellow oil (1, 10.5 g, 100%). ¹H NMR (CDCl₃, 400 MHz)δ 7.16 (t, J=7.4, 8.2 Hz, 1H), 6.82 (dd, J=1.0, 7.4 Hz, 1H), 6.70 (dd,J=1.1, 8.4 Hz, 1H), 5.14 (br s, 2H), 3.78 (s, 3H), 3.76 (s, 3H).

2-Ethyl 5-methyl 4-oxo-3,4-dihydroquinazoline-2,5-dicarboxylate (2) Amixture of 1 (2.1 g, 10 mmol) in acetic acid (10 mL), ethyl cyanoformate(1.0 g, 10 mmol), and 1 N HCl in acetic acid (10.5 mL) was heated to120° C. and stirred for 2.5 h. The reaction mixture was concentrated andthe crude residue was purified by flash chromatography (0.5% MeOH inCH₂Cl₂ to 1% MeOH in CH₂Cl₂) to afford a white solid (2, 2.35 g, 85%).¹H NMR (DMSO-d₆, 400 MHz) δ 7.91 (d, J=4.2 Hz, 2H), 7.59 (t, J=4.2 Hz,1H), 4.38 (q, J=7.1, 7.3, 14 Hz, 2H), 3.83 (s, 3H), 1.35 (t, J=7.3, 7.1Hz, 3H).

5-Methyl 2-hydroxymethyl-4-oxo-3,4-dihydroquinazoline-5-carboxylate (3)To a solution of 2 (2.0 g, 7.25 mmol) in EtOH (100 mL) was added NaBH₄(0.55 g, large excess) at 0° C. The resulting mixture was allowed towarm to room temperature and stirred for 6 h. The solvent was removedand EtOAc (50 mL) and H₂O (20 mL) were added. The aqueous layer wasextracted with EtOAc and CH₂Cl₂. The combined organic layers were dried(MgSO₄) and concentrated in vacuum. The crude residue was purified byflash chromatography (1% MeOH in CH₂Cl₂ to 5% MeOH in CH₂Cl₂) to afforda white solid (3, 1.0 g, 59%). ¹H NMR (CD₃OD, 400 MHz) δ 7.72 (t, J=7.4,8.2 Hz, 1H), 7.64 (d, J=8.4 Hz, 1H), 7.32 (d, J=7.1 Hz, 1H), 4.46 (s,2H), 3.82 (s, 3H).

5-Methyl3,4-dihydro-4-oxo-2-(dimethylamino)methyl-5-quinazoline-carboxylate (4a)To a suspension of 3 (130 mg, 0.55 mmol) in CH₂Cl₂ (5 mL) at roomtemperature was added methanesulfonyl chloride (47 uL, 0.61 mmol) underN₂. The reaction mixture was stirred overnight followed by addition ofdimethylamine hydrochloride (68 mg, 0.83 mmol) and Et₃N (168 mg, 1.67mmol) at room temperature. The resulting mixture was stirred overnight.The reaction mixture was concentrated in vacuum and purified by flashchromatography (1% MeOH in CH₂Cl₂ to 2.5% MeOH in CH₂Cl₂) to afford awhite solid (4a, 70 mg, 48%). ¹H NMR (CDCl₃, 400 MHz) δ 7.63-7.75 (m,2H), 7.36 (dd, J=1.9, 6.5 Hz, 1H), 3.94 (s, 3H), 3.46 (s, 2H), 2.31 (s,3H).

5-Methyl3,4-dihydro-4-oxo-2-(1-piperidinylmethyl)-5-quinazolinecarboxy-late (4b)To a solution of 3 (125 mg, 0.53 mmol) in CH₂Cl₂ (8 mL) was added Et₃N(81 mg, 0.80 mmol) and p-toluenesulfonyl chloride (826 mg, 0.59 mmol) at0° C. The reaction mixture was allowed to warm to room temperature andstirred for 2 h. The solvents were removed in vacuum. To the resultingoil was added EtOH (5 mL) and piperidine (54 mg, 0.64 mmol) at roomtemperature. The reaction mixture was heated at 50° C. for 1.5 h andthen cooled to room temperature. The solvent was removed in vacuum.EtOAc (40 mL) and H₂O (25 mL) were added and the aqueous layer waswashed with EtOAc (2×40 mL). The combined organic layers were washedwith brine, dried (Na₂SO₄), filtered and concentrated in vacuum. Thecrude residue was purified by flash chromatography (10% MeOH in CH₂Cl₂)to afford a light yellow oil (4b, 50 mg, 31%). ¹H NMR (CDCl₃, 400 MHz) δ7.74-7.65 (m, 2H), 7.43 (dd, J=1.8, 6.8 Hz, 1H), 5.28 (s, 2H), 3.95 (s,3H), 1.58-1.41 (m, 10H); MS (ES+): 302.03.

5-Methyl3,4-dihydro-4-oxo-2-(1-pyrrolidinylmethyl)-5-quinazolinecarboxy-late(4c) To a solution of 3 (125 mg, 0.53 mmol) in CH₂Cl₂ (8 mL) was addedEt₃N (81 mg, 0.80 mmol) and p-toluenesulfonyl chloride (826 mg, 0.59mmol) at 0° C. The reaction mixture was allowed to warm to roomtemperature and stirred for 2 h. The solvents were removed in vacuum. Tothe resulting oil was added EtOH (5 mL) and pyrrolidine (55 mg, 0.77mmol) at room temperature. The reaction mixture was heated at 50° C. for1.5 h and then cooled to room temperature. The solvent was removed invacuum. EtOAc (40 mL) and H₂O (25 mL) were added and the aqueous layerwas washed with EtOAc (2×40 mL). The combined organic layers were washedwith brine, dried (Na₂SO₄), filtered and concentrated in vacuum. Thecrude residue was purified by flash chromatography (10% MeOH in CH₂Cl₂)to afford a light yellow oil (4c, 40 mg, 26%) which was used directly innext step.

2,9-Dihydro-8-[(dimethylamino)methyl]-3H-pyridazino[3,4,5-de]quinazoline-3-one(5a) To a solution of 4a (70 mg, 0.27 mmol) in EtOH (4 mL) was addedNH₂NH₂ (2 mL) at room temperature. The resulting mixture was heated to100° C. and stirred overnight. The reaction mixture was concentrated invacuum and purified by flash chromatography (2% MeOH in CH₂Cl₂ with 0.5%NH₄OH to 4% MeOH in CH₂Cl₂ with 0.5% NH₄OH) to afford a yellow solid(5a, 30 mg, 45%). Mp>240° C. (dec.); ¹H NMR (CDCl₃, 400 MHz) δ 11.9 (brs, 1H), 7.78 (dd, J=0.76, 7.6 Hz, 1H), 7.67 (t, J=7.8 Hz, 1H), 7.48 (d,J=7.4 Hz, 1H), 3.42 (s, 2H), 2.43 (s, 6H); MS (ES−): 242.14; Anal. Calcdfor C₁₂H₁₃N₅O.(0.25H₂O): C, 58.17; H, 5.49; N, 28.27. Found: C, 57.91;H, 5.46; N, 28.55.

2,9-Dihydro-8-[(1-piperidinylmethyl]-3H-pyridazino[3,4,5-de]quinazoline-3-one(5b) To a solution of 4b (50 mg, 0.17 mmol) in EtOH (3 mL) was addedhydrazine (2.5 mL). The reaction mixture was stirred at 120° C. for 18 hand then cooled to room temperature. EtOAc (20 mL) and H₂O (20 mL) wasadded. The aqueous layer was washed with EtOAc. The combined organiclayers were washed with brine (10 mL), dried (Na₂SO₄). The crude residuewas purified by flash chromatography (10% MeOH in CH₂Cl₂) to afford awhite solid (5b, 23.6 mg, 49%). Mp: 250-252° C.; ¹H NMR (CDCl₃, 400 MHz)δ 10.01 (s, 1H), 7.80 (d, J=7.87 Hz, 1H), 7.50 (t, J=7.73, 1H), 7.45 (s,1H), 3.45 (s, 2H), 2.40-2.60 (m, 4H), 1.50-1.45 (m, 6H); MS (ES+): 284;Anal. Calcd for C₁₅H₁₇N₅O₁.(0.5H₂O): C, 61.63; H, 6.21; N, 23.96. Found:C, 61.53; H, 6.21; N, 23.81.

2,9-Dihydro-8-[(1-pyrrolidinylmethyl]-3H-pyridazino[3,4,5-de]quinazoline-3-one(5c) To a solution of 4c (40 mg, 0.14 mmol) in EtOH (5 mL) was addedhydrazine (2.5 mL). The reaction mixture was stirred at 120° C. for 18 hand then cooled to room temperature. EtOAc (20 mL) and H₂O (20 mL) wasadded. The aqueous layer was washed with EtOAc. The combined organiclayers were washed with brine (10 mL), dried (Na₂SO₄). The crude residuewas purified by flash chromatography (10% MeOH in CH₂Cl₂) to afford awhite solid (5c, 15 mg, 40%). Mp: 230-232° C.; ¹H NMR (CDCl₃, 400 MHz) δ10.70 (s, 1H), 8.25 (d, J=7.63 Hz, 1H), 8.10 (t, J=7.82 Hz, 1H),7.75-7.80 (m, 1H), 3.80 (s, 2H), 3.0 (s, 4H), 2.30 (s, 4H); MS (ES+):270.07; Anal. Calcd for C₁₄H₁₅N₅O₁.(0.23H₂O): C, 59.17; H, 5.39; N,24.25. Found: C, 59.16; H, 5.51; N, 24.39.

3-Oxo-2,9-dihydro-3H-pyridazino[3,4,5-de]quinazoline-8-carboxylichydrazide (6) A mixture of 2 (200 mg, 0.72 mmol) in NH₂NH₂ (3 mL) wasrefluxed overnight. The reaction mixture was cooled to room temperatureand the excess NH₂NH₂ was removed in vacuum. The crude residue waswashed with MeOH and CH₂Cl₂ to afford a yellow solid (6, 100 mg, 56%).Mp>300° C.; ¹H NMR (DMSO-d₆, 400 MHz) δ 11.93 (s, 1H), 9.94 (br s, 1H),7.76 (t, J=7.8 Hz, 1H), 7.64 (dd, J=0.94, 7.8 Hz, 1H), 7.59 (dd, J=0.94,7.8 Hz, 1H), 4.68 (br s, 2H); MS (ES+): 245.34; Anal. Calcd forC₁₀H₈N₆O₂.(0.8H₂O): C, 46.44; H, 3.74; N, 32.5. Found: C, 45.99; H,3.89; N, 32.89.

EXAMPLE 2 Preparation of8-Amino-2,9-dihydro-3H-pyridazino[3,4,5-de]quinazoline-3-one (9) (Scheme2)

Chloroformamidine hydrochloride (7) To a solution of cyanamide (1 g,23.78 mmol) in Et₂O (20 mL) was bubbling through HCl gas until no moreprecipitate formed. The precipitate was filtered and dried in vacuumover KOH to afford a white solid (7, 2.5 g, 93%) that was used directlyin the next step. MS (ES+): 116.12.

5-Methyl 2-amino-4-oxo-3,4-dihydroquinazoline-5-carboxylate (8) To asolution of 1 (2.75 g, 13 mmol) in Dowtherm A (25 mL) was added 7 (1.5g, 13 mmol) and Me₂SO₂ (7.96 g, 84.5 mmol) at room temperature. Thereaction mixture was heated to 165° C. and stirred for 2 h. The reactionmixture was cooled to room temperature and purified by flashchromatography (5% MeOH in CH₂Cl₂ to 10% MeOH in CH₂Cl₂) to afford awhite solid (8, 2.5 g, 88%). ¹H NMR (DMSO-d₆, 400 MHz) δ 11.05 (br s,1H), 7.56 (dd, J=7.5, 8.2 Hz, 1H), 7.25 (dd, J=0.95, 8.2 Hz, 1H), 6.98(d, J=7.2 Hz, 1H), 6.50 (br s, 2H), 3.76 (s, 3H).

8-Amino-2,9-dihydro-3H-pyridazino[3,4,5-de]quinazoline-3-one (9) Amixture of 8 (200 mg, 0.91 mmol) in EtOH/NH₂NH₂ (1:1, 10 mL) was heatedto 100° C. and stirred overnight. The reaction mixture was allowed tocool to room temperature. The resulting precipitate was filtered andwashed with EtOH to afford a yellow solid (9, 176 mg, 87%). Mp>300° C.;¹H NMR (DMSO-d₆, 400 MHz) δ11.40 (br s, 1H), 7.61 (t, J=7.8, 8.0 Hz,1H), 7.44 (dd, J=0.95, 8.0 Hz, 1H), 7.15 (dd, J=0.95, 7.8 Hz, 1H), 6.33(s, 2H); MS (ES+): 202.18; Anal. Calcd for C₉H₇N₅O.(1N₂H₄): C, 46.35; H,4.75; N, 42.04. Found: C, 46.2; H, 4.83; N, 41.76.

EXAMPLE 3 Preparation of2,9-Dihydro-3H-pyridazino[3,4,5-de]quinazoline-3-one (13) (Scheme 3)

5-Methyl 4-oxo-3,4-dihydroquinazoline-5-carboxylate (12) To a solutionof 1 (2.0 g, 9.57 mmol) in 2-methoxyethanol (30 mL) was addedformamidine acetate (2.5 g, 24.0 mmol) at room temperature. The reactionmixture was refluxed at 135° C. for 2 h. The reaction mixture was cooledto room temperature. The solvent was removed and water was added. Theresulting precipitate was filtered, washed with H₂O, and dried invacuum. The resulting off-white solid (12, 1 g, 50%) was used directlynext step. ¹H NMR (CD₃OD, 400 MHz) δ 8.01 (s, 1H), 7.72-7.80 (m, 1H),7.68 (dd, J=1.3, 1.1, 8.4, 8.2 Hz, 1H), 7.37 (dd, J=1.1, 1.3, 7.1, 7.2Hz, 1H), 3.83 (s, 3H).

2,9-Dihydro-3H-pyridazino[3,4,5-de]quinazoline-3-one (13) A mixture of12 (1.0 g, 5.0 mmol) in EtOH/NH₂NH₂ (1:1, 20 mL) was refluxed at 140° C.for 4 hrs. The reaction mixture was allowed to cool to room temperature.The solvent was removed partially and the resulting precipitate wasfiltered and washed with EtOH to afford a yellow solid (13, 400 mg,43%). Mp>300° C.; ¹H NMR (DMSO-d₆, 400 MHz) δ11.74 (br s, 1H), 7.77 (s,1H), 7.74 (t, J=7.8, 8.0 Hz, 1H), 7.62 (dd, J=0.96, 1.1, 7.8, 8.0 Hz,1H), 7.33 (dd, J=0.96, 7.8 Hz, 1H); MS (ES+): 187; Anal. Calcd forC₉H₆N₄O: C, 58.06; H, 3.25; N, 30.09. Found: C, 57.77; H, 3.32; N,30.29.

EXAMPLE 4 Preparation of9-[(Hydrozinooxy)carbonyl]-2,7-dihydro-6-methoxy3H-pyrido-[4,3,2-de]phthalazin-3-one (16) (Scheme 4)

Diethyl[[[2-methoxy-5-(methoxycarbonyl)phenyl]amino]methylene]propanedioate(14) A mixture of methyl 3-amino-4-methoxybenzoate (2.0 g, 11 mmol) anddiethyl ethoxymethylenemalonate (5 mL) was stirred at room temperaturefor 5 h. Water was added and the precipitate was filtered, washed withH₂O and Et₂O, dried in vacuum to afford a yellow solid (14, 3.9 g,100%). ¹H NMR (DMSO-d₆, 400 MHz) δ 11.98 (d, J=14 Hz, 1H), 8.51 (d, J=14Hz, 1H), 7.89 (s, 1H), 7.75 (dd, J=1.7, 8.6 Hz, 1H), 7.25 (d, J=8.6 Hz,1H), 4.20 (q, J=7.1, 14 Hz, 2H), 4.14 (q, J=7.1, 14 Hz, 2H), 3.98 (s,3H), 3.84 (s, 3H), 1.20-1.29 (m, 6H).

3-Ethyl 5-methyl 1,4-dihydro-8-methoxy-4-oxo-3,5-quinolinedioate (15) Asuspension of 14 (1.0 g, 2.85 mmol) in Dowtherm A (10 mL) was heated at300˜360° C. for 1 h. The reaction mixture was cooled to roomtemperature. The resulting precipitate was filtered, washed with Et₂O,and dried in vacuum to afford a yellow solid (15, 1.3 g, 46%). ¹H NMR(DMSO-d₆, 400 MHz) δ 12.09 (br s, 1H), 8.35 (s, 1H), 7.29 (q, J=8.2, 20Hz, 2H), 4.20 (q, J=7.2, 14, 7.1 Hz, 2H), 4.02 (s, 3H), 3.77 (s, 3H),1.25 (t, J=7.1, 7.2 Hz, 3H).

9-[(Hydrozinooxy)carbonyl]-2,7-dihydro-6-methoxy3H-pyrido-[4,3,2-de]phthalazin-3-one (16) A mixture of 15 (400 mg, 1.31mmol) in EtOH/NH₂NH₂ (1:1, 10 mL) was refluxed at 140° C. for 3 hrs. Thereaction mixture was allowed to cool to room temperature. The resultingprecipitate was filtered and washed with Et₂O to afford a yellow solid(16, 80 mg, 22%). Mp>300° C.; ¹H NMR (DMSO-d₆, 300 MHz) 11.83 (s, 1H),10.24 (s, 1H), 10.07 (s, 1H), 7.97 (s, 1H), 7.73 (d, J=8.5 Hz, 1H), 7.55(d, J=8.4 Hz, 1H), 4.57 (d, J=3.8 Hz, 2H), 4.04 (s, 3H); Anal. Calcd forC₁₂H₁₁N₅O₃.(1H₂O): C, 49.48; H, 4.5; N, 24.04. Found: C, 49.44; H, 4.53;N, 24.17.

EXAMPLE 5 Preparation of9-[(Hydrozinooxy)carbonyl]-2,7-dihydro-3H-pyrido-[4,3,2-de]phthalazin-3-one(21) (Scheme 5)

Methyl 4-chloro-3-aminobenzoate (17) To a solution of methyl4-chloro-3-nitrobenzoate (3.0 g, 13.9 mmol) in EtOAc (50 mL) was added10% Pd/C (0.64 g). The resulting mixture was hydrogenated under H₂ (50psi) on a Parr hydrogenation apparatus at room temperature for 1 h. Thecatalyst was filtered off on a pad of celite and the filtrate wasconcentrated in vacuum to afford a dark oil (17, 2.0 g, 77%). ¹H NMR(DMSO-d₆, 300 MHz) δ 7.40 (d, J=2.1 Hz, 1H), 7.30 (d, J=8.2 Hz, 1H),7.08 (dd, J=2.1, 8.4 Hz, 1H), 3.79 (s, 3H).

Diethyl[[[2-chloro-5-(methoxycarbonyl)phenyl]amino]methylene]propanedioate(18) A mixture of 17 (2.0 g, 10.7 mmol) and diethylethoxymethylene-malonate (5 mL) was stirred at room temperatureovernight. Water was added and the resulting precipitate was filtered,washed with H₂O and Et₂O, dried in vacuum to afford a yellow solid (18,3.0 g, 79%). ¹H NMR (DMSO-d₆, 300 MHz) δ 11.20 (d, J=13 Hz, 1H), 8.54(d, J=8.4 Hz, 1H), 8.05 (d, J=1.3 Hz, 1H), 7.73 (d, J=8.2 Hz, 1H), 7.69(dd, J=1.7, 8.4 Hz, 1H), 4.22 (q, J=7.1, 14 Hz, 2H), 4.16 (q, J=7.1, 14Hz, 2H), 3.88 (s, 3H), 1.25 (t, J=7.1 Hz, 3H), 1.25 (t, J=7.1 Hz, 3H).

9-[(Hydrozinooxy)carbonyl]-2,7-dihydro-6-chloro-3H-pyrido-[4,3,2-de]phthalazin-3-one(20) A suspension of 14 (1.0 g, 2.81 mmol) in Dowtherm A (10 mL) washeated at 300˜330° C. for 45 min. The reaction mixture was cooled toroom temperature. Most of the solvent was removed in vacuum. Thesolution was used directly next step.

To the above solution was added EtOH/NH₂NH₂ (1:1, 10 mL) at roomtemperature and the reaction mixture was refluxed at 140° C. overnight.After the reaction, the reaction mixture was allowed to cool to roomtemperature. The resulting precipitate was filtered and washed with Et₂Oto afford a yellow solid (20, 220 mg, 28%). Mp>300° C.; ¹H NMR (CDCl₃,300 MHz) δ 10.05 (s, 1H), 8.12 (s, 1H), 7.86 (d, J=8.4 Hz, 1H), 7.58 (d,J=8.2 Hz, 1H), 4.55 (s, 2H); Anal. Calcd for C₁₁H₈ClN₅O₂: C, 46.09; H,3.16; N, 24.43; Cl, 12.37. Found: C, 45.79; H, 3.35; N, 24.88; Cl,12.25.

9-[(Hydrozinooxy)carbonyl]-2,7-dihydro-3H-pyrido-[4,3,2-de]phthalazin-3-one(21) To a suspension of 20 (110 mg, 0.40 mmol) in DMF (50 mL) was added10% Pd/C (100 mg). The resulting mixture was hydrogenated under H₂ (40psi) on a Parr hydrogenation apparatus at room temperature overnight.The catalyst was filtered off on a pad of celite and washed with Et₂O.The filtrate was concentrated in vacuum to afford a yellow solid (21, 20mg, 21%). Mp>300° C.; ¹H NMR (DMSO-d₆, 300 MHz) δ 12.17 (s, 1H), 11.77(d, J=5.7 Hz, 1H), 10.54 (s, 1H), 8.17 (d, J=6.3 Hz, 1H), 7.82 (t, J=7.8Hz, 1H), 7.73 (d, J=7.6 Hz, 1H), 7.55 (d, J=8.2 Hz, 1H); Anal. Calcd forC₁₁H₉N₅O₂.(1.7H₂O): C, 48.25; H, 4.56; N, 25.57. Found: C, 48.44; H,4.06; N, 25.12.

EXAMPLE 6 Preparation of Phthalazine Derivatives (27, 28 and 30) (Scheme6)

Methyl 4-chloro-3-amino-benzoate (22). To a solution of4-chloro-3-amino-benzoic acid (10.0 g, 58.3 mmol) in methanol (50 mL)was added acetyl chloride (13.0 mL) dropwise at room temperature. Afterthe addition, the reaction mixture was heated at 70° C. for 3 h and thencooled to room temperature. The solvent was removed under reducedpressure and water (100 mL) was added. The resulting solution wasneutralized with NaHCO₃ followed by extraction with EtOAc (3×100 mL).The combined organic layers were dried (MgSO₄) and concentrated invacuum to afford a yellow oil (22, 10.5 g, 97%).

¹H NMR (DMSO-d₆, 300 MHz) δ 7.41 (??d, J=2.1 Hz, 1H), 7.31 (d, J=8.4 Hz,1H), 7.09 (dd, J=2.1, 8.4 Hz, 1H), 5.66 (br s, 2H), 3.80 (s, 3H).

2-[2-Chloro-5-(methoxycarbonyl)phenylamino]-2-butenedioic acid diethylester (23). To a solution of 22 (5.00 g, 26.9 mmol) in ethanol (50 mL)was added but-2-ynedioic acid diethyl ester (5.02 g, 29.5 mmol) at roomtemperature. The reaction mixture was heated at 90° C. for 18 h and thencooled to room temperature. The solvent was removed under reducedpressure. The crude residue was washed with a solution of 1:1Hexanes/diethyl ether (100 mL) to afford a white solid (23, 9.08 g,95.1%). ¹H NMR (DMSO-d₆, 300 MHz) δ ?9.80 (s, 1H), 7.65-7.66 (m, 2H),7.38 (s, 1H), 5.54 (s, 1H), 4.13-4.22 (m, 4H), 3.83 (s, 3H), 1.22 (t,J=7.1 Hz, 3H), 1.11 (t, J=7.1 Hz, 3H).

8-Chloro-4-oxo-1,4-dihydro-2,5-quinolinedicarboxylic acid 2-ethyl5-methyl ester (24). A suspension of 23 (9.00 g, 25.4 mmol) in biphenylether (100 mL) was heated to 250° C. and stirred for 3 h. Then thereaction mixture was cooled to room temperature and purified by flashchromatography (hexanes to EtOAc) to afford a colorless oil (24, 3.88 g,49.4%). ¹H NMR (CDCl₃, 300 MHz) δ 9.39 (br s, 1H), 7.75 (d, J=8.0 Hz,1H), 7.24 (d, J=8.0 Hz, 1H), 6.96 (s, 1H), 4.54 (q, J=7.0 Hz, 2H), 4.00(s, 3H), 1.46 (t, J=7.0 Hz, 3H).

8-Chloro-4-oxo-1,4-dihydro-2,5-quinolinedicarboxylic acid (25). To asolution of 24 (10.0 g, 32.3 mmol) in ethanol (100 mL) was added 10%aqueous KOH solution (100 mL) at room temperature. The reaction mixturewas heated at 100° C. for 24 h and then cooled to room temperature. Thereaction mixture was concentrated in vacuum. The resulting aqueoussolution was acidified with 2N HCl to pH=6.5, where a solid precipitatedand was collected through a filtration. The solid was washed with H₂O(100 mL) and Et₂O (100 mL), and dried in vacuum at 60° C. to give anoff-white solid (25, 8.00 g, 92.9%). ¹H NMR (DMSO-d₆, 300 MHz) δ ?7.97(d, J=7.8 Hz, 1H), 7.39 (d, J=7.8 Hz, 1H), 6.90 (s, 1H).

4-Oxo-1,4-dihydro-2,5-quinolinedicarboxylic acid (26). To a solution of25 (7.90 g, 29.5 mmol) in 10% aqueous KOH solution (100 mL) was added10% Pd/C (0.500 g). The reaction mixture was hydrogenated (50 psi) on aParr hydrogenation apparatus at room temperature for 4 h. The catalystwas filtered off on a pad of celite and the filtrate was acidified with2N HCl to pH=6.5, where a solid precipitated and was collected through afiltration. The solid was washed with H₂O (100 mL) and Et₂O (100 mL),and dried in vacuum at 60° C. to give an off-white solid (26, 6.79 g,98.6%). ¹H NMR (DMSO-d₆, 300 MHz) δ ?8.12 (d, J=8.2 Hz, 1H), 7.76 (t,J=8.2 Hz, 1H), 7.48 (d, J=8.2 Hz, 1H), 6.74 (s, 1H).

3-Oxo-2,7-dihydro-3H-pyrido[4,3,2-de]phthalazine-8-carboxylic acid (27).To a solution of 26 (23.1 g, 99.1 mmol) in ethylene glycol (300 mL)heated to 140° C. was carefully added hydrazine hydrate (4.75 g, 95.0mmol) dropwise. The reaction mixture was heated to 150° C. and stirredfor 44 h. Removal of the 90% of the ethylene glycol under vacuum gave aprecipitate, which was washed with methanol (100 mL) and diethyl ether(100 mL) to give a yellow solid (27, 16.5 g, 72.7%). ¹H NMR (DMSO-d₆,300 MHz) δ 11.85 (s, 1H), 10.43 (s, 1H), 7.61-7.63 (m, 2H), 7.43-7.46(m, 1H), 6.49 (s, 1H).

3-Oxo-2,7-dihydro-3H-pyrido[4,3,2-de]phthalazine-8-carboxylic acidsodium salt (28) To a solution of NaOH (1 g) in water (100 mL) was added27 (1.00 g, 4.36 mmol) and the reaction mixture was stirred for 1 h. Anyinsoluble material was filtered off through a fine frit glass funnel andthe aqueous layer was concentratedin vacuum to afford a yellow solid(28, 0.898 g, 82.1%). ¹H NMR (D₂O, 300 MHz) δ ?7.23 (t, J=7.8 Hz, 1H),6.97 (d, J=7.8 Hz, 1H), 6.88 (d, J=7.8 Hz, 1H), 5.91 (s, 1H). Anal:Calcd for C₁₁H₆N₃O₄Na (10H₂O and 2.45NaOH): C, 35.98; H, 2.87; N, 11.44.Found: C, 35.76; H, 2.86; N, 11.82.

4-Oxo-1,4-dihydro-2,5-quinolinedicarboxylic acid 2-ethyl 5-methyl ester(29). To a suspension of 24 (4.50 g, 14.5 mmol) in EtOAc (100 mL) wasadded Et₃N (50 mL). The reaction mixture was heated to 50° C. togenerate a clear solution. 10% Pd/C (0.500 g) was added carefully. Thereaction mixture was hydrogenated (50 psi) on a Parr hydrogenationapparatus at room temperature for 4 h. Then the catalyst was filteredoff on a pad of celite and the filtrate washed with water (100 mL). Theaqueous layer was extracted with EtOAc (100 mL) and the combined organiclayers were dried (MgSO₄), filtered, and concentrated. The crude residuewas washed with Et₂O (100 mL) to give an off-white solid (29, 3.46 g,99.1%). ¹H NMR (DMSO-d₆, 300 MHz) δ ?8.05 (d, J=8.6 Hz, 1H), 7.75 (t,J=8.6 Hz, 1H), 7.29 (d, J=8.6 Hz, 1H), 6.63 (s, 1H), 4.44 (q, J=7.3 Hz,2H), 3.80 (s, 3H), 1.37 (t, J=7.3 Hz, 3H).

3-Oxo-2,7-dihydro-3H-pyrido[4,3,2-de]phthalazine-8-carboxylic acidhydrazide (30). To a solution of 29 (1.38 g, 5.02 mmol) in ethyleneglycol (25 mL) was added hydrazine hydrate (1.00 g, 20.0 mmol). Thereaction mixture was heated to 180° C. and stirred for 18 h. Aftercooled to room temperature, the resulting yellow precipitate wasfiltered, washed with Et₂O (100 mL), dried in vacuum at 90° C. for 6 hto give a yellow solid (30, 1.10 g, 90.2%). ¹H NMR (DMSO-d₆, 300 MHz) δ11.89 (s, 1H), 10.51 (s, 1H), 10.09 (s, 1H), 7.62-7.68 (m, 2H),7.46-7.49 (m, 1H), 6.57 (s, 1H), 4.60 (br s, 2H); Anal. Calcd forC₁₁H₉N₅O₂: C, 54.32; H, 3.73; N, 28.79. Found: C, 54.42; H, 3.72; N,28.86.

EXAMPLE 7 Preparation of6-Fluoro-3-oxo-2,7-dihydro-3H-pyrido[4,3,2-de]phthalazine-8-carboxylicacid (36) (Scheme 7)

Methyl 4-fluoro-3-nitrobenzoate(31). To a solution of4-fluoro-3-nitrobenzoic acid (15.0 g, 81.1 mmol) in MeOH (500 mL) wasadded H₂SO₄ (1 mL) and the reaction mixture was heated at 70° C. for 72h and then cooled to room temperature. The reaction solution was dneutralized to pH=7.0 with NaHCO₃, then concentrated in vacuum. H₂O (300mL) was added and washed with EtOAc (3×300 mL). The combined organiclayers were dried (MgSO₄) and concentrated in vacuum to give a yellowoil (31, 16.0 g, 99.0%). ¹H NMR (CDCl₃, 300 MHz) δ 8.76 (dd, J=2.3, 7.3Hz, 1H) 8.30-8.34 (m, 1H), 7.39 (t, J=10.1 Hz, 1H), 3.98 (s, 3H).

Methyl 4-fluoro-3-aminobenzoate (32) To a solution of 31 (16.0 g, 80.3mmol) in MeOH (250 mL) was added 10% Pd/C (0.500 g). The reactionmixture was hydrogenated (50 psi) on a Parr hydrogenation apparatus atroom temperature for 4 h. The catalyst was filtered off on a pad ofcelite and the filtrate was concentrated in vacuum to afford a colorlessoil (32, 12.2 g, 90.0%). ¹H NMR (CDCl₃, 300 MHz) δ ?7.48 (dd, J=2.1, 8.6Hz, 1H), 7.39-7.43 (m, 1H), 7.02 (dd, J=8.6, 10.7 Hz, 1H), 3.88 (s, 3H),3.84 (br s, 2H).

2-[2-Fluoro-5-(methoxycaronyl)phenylamino]-2-butenedioic acid diethylester (33) To a solution of 32 (5.00 g, 29.6 mmol) in ethanol (200 mL)was added but-2-ynedioic acid diethyl ester (6.29 g, 37.0 mmol) and thereaction mixture was stirred at room temperature for 24 h. The solventwas then removed under reduced pressure and the residue was purified byflash chromatography (Hexanes to 25% EtOAc in Hexanes) to give a yellowoil (33, 7.63 g, 76.5%). ¹H NMR (CDCl₃, 300 MHz) δ 9.64 (br s, 1H),7.72-7.78 (m, 1H), 7.62 (dd, J=7.8, 1.9 Hz, 1H), 7.12 (t, J=10.3 Hz,1H), 5.61 (s, 1H), 4.19-4.27 (m, 4H), 3.89 (s, 3H), 1.31 (t, J=7.0, 3H),1.20 (t, J=7.0, 3H).

8-Fluoro-4-oxo-1,4-dihydro-2,5-quinolinedicarboxylic acid 2-ethyl5-methyl ester (34). A suspension of 33 (25.0 g, 73.7 mmol) in DowthermA (200 mL) was heated to 300° C. over the course of 30 minutes and thenstirred at 300° C. for 1 h. The reaction solution was cooled to roomtemperature and purified by flash chromatography (Hexanes to EtOAc) toafford a white solid (34, 12.1 g, 56.1%). ¹H NMR (CDCl₃, 300 MHz) δ 9.19(br s, 1H), 7.42 (t, J=8.2, 1H), 7.21-7.25 (m, 1H), 6.92 (s, 1H), 4.51(q, J=7.1, 2H), 3.99 (s, 3H), 1.46 (t, J=7.1, 3H).

8-Fluoro-4-oxo-1,4-dihydro-2,5-quinolinedicarboxylic acid (35). To a 2 Naqueous HCl (200 mL) solution was added 34 (10.0 g, 34.1 mmol). Thereaction mixture was heated at 100° C. for 18 h and then cooled to roomtemperature. The resulting precipitate was filtered and dried undervacuum at 60° C. for 18 h to afford an off-white solid (35, 8.11 g,94.7%). ¹H NMR (DMSO-d₆, 300 MHz) δ ?7.65 (t, J=8.2, 1H), 7.39-7.43 (m,1H), 6.93 (s, 1H).

6-Fluoro-3-oxo-2,7-dihydro-3H-pyrido[4,3,2-de]phthalazine-8-carboxylicacid (36). To a solution of 35 (0.600 g, 2.39 mmol) in EtOH (5 mL) wasadded hydrazine (0.0840 g, 2.63 mmol). The resulting mixture was heatedat 90° C. for 24 hand then cooled to room temperature. The resultingprecipitate was filtered, washed with Et₂O (25 mL), and dried in vacuumat 60° C. for 6 h to give a white solid (36, 0.484 g, 74.5%). ¹H NMR(DMSO-d₆, 300 MHz) δ ?8.25-8.30 (m, 1H), 7.51-7.57 (m, 1H), 7.06 (s,1H); Anal. Calcd for C₁₁H₆N₃O₃F (2H₂O): C, 46.65; H, 3.56; N, 14.84.Found: C, 46.80; H, 3.59; N, 14.86.

EXAMPLE 8 Preparation of6-Methoxy-3-oxo-2,7-dihydro-3H-pyrido[4,3,2-de]phthalazine-8-carboxylicacid hydozide (39) (Scheme 8)

2-[2-Methoxy-5-(methoxycarbonyl)phenylamino]-2-butenedioic acid dimethylester (37) To a solution of Methyl 3-amino-4-methoxybenzoate (5.0 g,27.6 mmol) in MeOH (60 mL) was added dimethyl acetylenedicarboxylate(4.31 g, 30.36 mmol) at room temperature. The reaction mixture wasrefluxed for 20 min and then cooled to room temperature. The solvent wasremoved. The crude residue was triturated in ether to give a yellowsolid (37, 4.35 g, 49%). ¹H NMR (DMSO-d₄, 400 MHz) δ 9.8 (s, 1H), 7.90(dd, J=1.9 Hz, 8.6 Hz, 1H), 7.50-7.60 (m, 1H), 7.35 (d, J=8.59 Hz, 1H),5.50 (s, 1H), 4.08 (s, 3H), 4.0 (s, 3H), 3.90 (s, 3H), 3.85 (s, 3H).

8-Methoxy-4-oxo-1,4-dihydro-2,5-quinolinedicarboxylic acid dimethylester (38) A suspension of 37 (4.20 g, 12.99 mmol) in Dowtherm A (30 mL)was heated at 300° C. for 35 min while monitored by TLC and then cooledto 0° C. The resulting precipitate was filtered and washed with Et₂O toafford a white solid (37, 2.0 g, 53%). ¹H NMR (DMSO-d₆, 400 MHz) δ 10.0(s, 1H), 7.34 (d, J=7.84 Hz, 1H), 7.30 (d, J=8.09 Hz, 1H), 6.60 (s, 1H),4.05 (s, 3H), 3.96 (s, 3H), 3.77 (s, 3H).

6-Methoxy-3-oxo-2,7-dihydro-3H-pyrido[4,3,2-de]phthalazine-8-carboxylicacid hydrozide (39) To a suspension of 38 (0.45 g, 1.54 mmol) in EtOH(20 mL) was added hydrazine (1 mL) at room temperature. The reactionmixture was heated at 68° C. for 1.5 h and then cooled to roomtemperature. The resulting precipitate was filtered and washed with Et₂Oto afford a white solid (39, 0.31 g, 74%). ¹H NMR (DMSO-d₆, 300 MHz) δ10.5 (br s, 1H), 9.0 (br s, 1H), 7.15-7.25 (m, 2H), 7.1 (br s, 1H), 6.8(br s, 1H), 4.90 (br s, 2H), 4.03 (s, 3H). Mp: 305-307° C.; Anal. Calcdfor C₁₂H₁₁N₅O₃.(0.75H₂O): C, 50.26; H, 4.39; N, 24.42. Found: C, 50.33;H, 4.46; N, 22.04.

EXAMPLE 9 Preparation of diazabenzo[de]anthracen-3-one Derivatives 43-48(Scheme 9)

7-bromomethyl-9-oxoxanthene-1-carboxylic acid methyl ester (41).Brominating agents include N-bromosuccinimide, bromine, complexedbromine such as pyridinium bromide, and the like can be used to convert7-methyl-9-oxoxanthene-1-carboxylic acid methyl ester 40 to7-bromomethyl-9-oxoxanthene-1-carboxylic acid methyl ester 41. Solventsinclude chlorinated hydrocarbons, dipolar aprotic solvents, and variousethers. Temperature can range from 0-100 C. For example, a suspension of7-methyl-9-oxoxanthene-1-carboxylic acid methyl ester (0.1 mol),N-bromosuccinimide (0.12 Mol) and benzoylperoxide (10 mg) in dry carbontetrachloride (300 mL) is stirred at 60 C for 6 hours. The mixture isfiltered, and the solid is washed successively with small amounts ofchloroform, water and ether, and then dried to leave a desired productas white solid. For example, to a solution of compound 40 (1.97 g, 7.4mmol) in carbon tetrachloride (400 mL) was added N-bromosuccinimide(1.44 g, 8.1 mmol) and a catalytic amount of benzoyl peroxide (45 mg,3%). The reaction mixture was heated to reflux for 6 h and cooled toroom temperature. The white precipitate was filtered out. The solventswere removed and the residue was recrystallized from EtOAc and hexanes.The crude product (2.05 g) was obtained and further recrystallizationgave a pure white solid product 41 (1.15 g, 45%). ¹H NMR (400 MHz,CDCl₃) of 2: 8.27 (d, J=2.5 Hz, 1H), 7.80-7.72 (m, 2H), 7.57 (dd, J=8.5and 1.1 Hz, 1H), 7.48 (d, J=8.5 Hz, 1H), 7.33 (dd, J=7.0 and 1.1 Hz,1H), 4.57 (s, 2H), 4.00 (s, 3H).

Displacement of the bromo group of compound 41 with nucleophiles such asamine using General procedure A provides the compound 42. The ketoester42 can be cyclized with hydrazine using general procedure B to givedesired final products. Compounds 43-48.

General Procedure A

To a solution of the bromo compound (41, 10 mmol) in dry DMF (100 mL) isadded potassium carbonate (100 mmol) and secondary amine (10 mmol). Thereaction mixture is heated to 70 C for 6 hours and cooled to roomtemperature. Water (100 mL) is added to the reaction mixture, followedby ethyl acetate (200 mL). The organic layer is collected, washed withwater, brine and dried over sodium sulfate. The solvents are removed invacuo. The residue is purified by column chromatography on silica gelusing ethyl acetate/hexanes as eluent to give the product 42 in 50-90%of yield. For example, a procedure to make the intermediate toward tosynthesize compound 43: To a solution of compound 41 (1.53 g, 4.4 mmol)in CH₃CN (50 mL) was added K₂CO₃ (1.2 g, 8.7 mmol) and 1-methypiperazine(0.51 mL, 4.6 mmol). The reaction mixture was heated to reflux overnightand cooled to room temperature. The solid was filtered and the solventswere evaporated. The residue was workup by ethyl acetate and water bynormal procedure. Column chromatography or acid/base extraction (theresidue were added 150 mL of 1N HCl and 150 mL of EtOAc. The aq layerwas separated and washed by 100 mL of EtOAC. Then the aq. layer wasadded 6N NaOH until pH>9. The solution was extracted by 100 mL of EtOActwice. Organic layers were combined, washed by water and brine, driedover MgSO₄. Solvents were evaporated to give an oil 42a (0.95 g, 59%).¹H NMR (400 MHz, CDCl₃) of 42a: 8.18 (d, J=2.5 Hz, 1H), 7.75-7.71 (m,2H), 7.56 (dd, J=8.5 and 1.1 Hz, 1H), 7.45 (d, J=8.5 Hz, 1H), 7.32 (dd,J=7.0 and 1.1 Hz, 1H), 4.04 (s, 3H), 3.58 (s, 2H), 2.45 (br, 8H), 2.27(s, 3H).

General Procedure B

A benzopyrano[4,3,2-de]phthalazine ring can be formed by condensation ofthe ketone easter with hydrazine. To a solution of the ketonester 42 (5mmol) in absolute ethanol (10 mL) is added anhydrous hydrazine inethanol (1 mL) drop wise at room temperature. The Solution is refluxedfor overnight and cooled to room temperature. Ice-cold water (100 mL) isadded and white solid is separated. The solid is collected by vacuumfiltration and washed with water and small amount of methanol to give awhite solid product in 40-85% of yield.

10-(4-Methyl-piperazin-1-ylmethyl)-2H-7-oxa-1,2-diaza-benzo[de]anthracen-3-one(43).

To a solution of compound 42a (0.91 g, 2.5 mmol) in EtOH (15 mL) whenrefluxing was added hydrazine monohydrate (2 mL, 41 mmol). The reactionmixture was stirred for 5 h and cooled. Solvents were removed and waterwas added. Precipitate was filtered out, washed with 15% EtOH andcollected to afford a white solid (0.85 g, 98%) 43. MS (ES+): 349; ¹HNMR (400 MHz, DMSO-d₆): 12.61 (s, 1H), 7.99 (d, J=2.0 Hz, 1H), 7.92-7.85(m, 2H), 7.68 (dd, J=7.5 and 2.0 Hz, 1H), 7.45 (dd, J=8.5 and 2.0 Hz,1H), 7.35 (d, J=8.5 Hz, 1H), 3.52 (s, 2H), 2.40 (bs, 8H), 2.18 (s, 3H).Anal. Calcd. for C₂₀H₂₀N₄O₂: C, 68.95; H, 5.79; N, 16.08. Found: C,69.04; H, 5.75; N, 16.2.

10-(4-Ethyl-piperazin-1-ylmethyl)-2H-7-oxa-1,2-diaza-benzo[de]anthracen-3-one(44).

Prepared from the compound 41 and 1-ethylpiperazine according to GeneralProcedure A and B. Purification of compound by crystallization inethanol gave a white solid product 44. MS (ES+): 363; ¹H-NMR (DMSO-d₆,400 MHz): 12.62 (s, 1H), 8.01 (s, 1H), 7.93-7.86 (m, 2H), 7.70 (dd,J=7.0 and 2.0 Hz, 1H), 7.47 (dd, J=8.5 and 2.0 Hz, 1H), 7.37 (d, J=8.5Hz, 1H), 3.55 (s, 2H), 2.52-2.30 (b, 10H), 1.04 (m, 3H). Anal. Calcd.for C₂₁H₂₂N₄O₂.(0.3H₂O): C, 68.57; H, 6.19; N, 15.23. Found: C, 68.21;H, 6.19; N, 15.38

10-[4-(2-Hydroxy-ethyl)-piperazin-1-ylmethyl]-2H-7-oxa-1,2-diaza-benzo[de]anthracen-3-one(45).

Prepared from the compound 41 and N-(2-hydroxyethyl)piperazine accordingto General Procedure A and B. Purification of compound bycrystallization in ethanol gave a white solid product 45. MS (ES+): 379;¹H-NMR (DMSO-d₆, 400 MHz): 12.62 (s, 1H), 8.00 (s, 1H), 7.92-7.85 (m,2H), 7.69 (d, J=7.0 Hz, 1H), 7.42 (d, J=8.5 Hz, 1H), 7.31(d, J=8.6 Hz,1H), 3.53-3.48 (m, 4H), 2.50-2.35 (m, 10H). Anal. Calcd. for C₂₁H₂₂N₄O₃:C, 66.65; H, 5.86; N, 14.81. Found: C, 66.39; H, 5.80; N, 14.94.

10-{4-[2-(2-Hydroxy-ethoxy)-ethyl]-piperazin-1-ylmethyl}-2H-7-oxa-1,2-diazabenzo[de]anthracen-3-one(46). Prepared from the compound 41 and 1-hydroxyethylethoxypiperazineaccording to General Procedure A and B. Purification of compound bycrystallization in ethanol gave a white solid product 7. MS (ES+): 423.¹H-NMR (DMSO-d₆, 400 MHz): 12.62 (s, 1H), 8.01 (s, 1H), 7.93-7.95 (m,2H), 7.69 (dd, J=7.0 and 2.0 Hz, 1H), 7.47 (dd, J=8.5 and 2.0 Hz, 1H),7.37 (d, J=8.5 Hz, 1H), 3.57-3.50 (m, 4H), 3.47 (m, 2H), 3.39 (m, 2H),2.49-37 (m, 10H). Anal. Calcd. for C₂₃H₂₆N₄O₄.(0.15H₂O): C, 64.97; H,6.23; N, 13.18. Found: C, 64.95; H, 6.21; N, 13.24.

10-{[Bis-(2-hydroxy-ethyl)-amino]-methyl}-2H-7-oxa-1,2-diaza-benzo[de]anthracen-3-one(47)

Prepared from the compound 41 and diethanolamine according to GeneralProcedure A and B. Purification of compound by crystallization inethanol gave a white solid product 47. MS (ES+): 354; Mp 222-225 C;¹H-NMR (DMSO-d₆, 400 MHz): 12.59 (s, 1H), 8.00 (s, 1H), 7.91-7.84 (m,2H), 7.67 (dd, J=7.0 and 2.0 Hz, 1H), 7.52 (dd, J=8.5 and 2.0 Hz, 1H),7.34 (d, J=8.5 Hz, 1H), 3.70 (s, 2H), 3.47 (t, J=6.4 Hz, 4H), 2.56 (t,J=6.4 Hz, 4H). Anal. Calcd. for C₁₉H₁₉N₃O₄: C, 64.58; H, 5.42; N, 11.89.Found: C, 64.32; H, 5.44; N, 11.90.

10-(4-Pyrrolidin-1-yl-piperidin-1-ylmethyl)-2H-7-oxa-1,2-diaza-benzo[de]anthracen-3-one(48)

Prepared from the compound 41 and 4-(1-pyrrolidinyl)piperidine accordingto General Procedure A and B. Purification of compound bycrystallization in ethanol gave a white solid product 48. MS (ES+): 403;Mp 258-264 C; ¹H-NMR (CDCl₃, 400 MHz): 9.93 (s, 1H), 8.06 (m, 2H), 7.81(t, J=8.0 Hz, 1H), 7.55 (dd, J=8.0, 1.0 Hz, 1H), 7.50 (dd, J=8.0, 2.5Hz, 1H), 7.26 (d, J=8.0 Hz, 1H), 3.55 (s, 2H), 2.90 (m, 2H), 2.61 (m,4H), 2.06 (m, 2H), 1.87-1.62 (m, 9H). Anal. Calcd. forC₂₄H₂₆N₄O₂.(0.5H₂O): C, 70.05; H, 6.61; N, 13.62. Found: C, 70.25; H,6.42; N, 13.59.

EXAMPLE 10 Preparation of diazabenzo[de]anthracen-3-one Derivatives49-55 (Scheme 10)

10-Aminomethyl-2H-7-oxa-1,2-diaza-benzo[de]anthracen-3-one (49). To asolution of 7-bromomethyl-9-oxoxoanthese-1-carboxylic acid methyl ester41 (0.70 g, 2.0 mmol) in anhydrous DMF (10 mL) was added Potassiumphthalimide (0.44 g, 2.4 mmol). The reaction mixture was stirred underreflux for 3 hours, then cooled and the solvent was removed in vacuo. Tothe residue was added 100 mL of ethyl acetate and washed with water andbrine, then dried with MgSO₄. The solvent was removed in vacuo to afforda white solid (0.53 g, 63%). The white solid (0.53 g) was dissolved inhydrazine (5 mL) and ethanol (10 mL) solution. The reaction mixture wasstirred under reflux for 3 hours. Solution was concentrated in vacuo. Tothe concentrated solution was added water. A white precipitate was formand filtered out. The white solid was dried to afford compound 49 (0.30g, 87%). MS (FAB (M+1)): 266; ¹H-NMR (DMSO-d₆, 400 MHz): 12.60 (sb, 1H),8.04 (d, J=2.0 Hz, 1H), 7.92-7.80 (m, 2H), 7.68 (dd, J=7.5 and 2.0 Hz,1H), 7.50 (dd, J=8.5 and 2.0 Hz, 1H), 7.34 (d, J=8.5 Hz, 1H), 3.76 (s,2H); Anal. Calcd. for C₁₅H₁₁N₃O₂: C, 67.92; H, 4.18; N, 15.84. Found: C,67.67; H, 4.20; N, 15.64.

N-(3-Oxo-2,3-dihydro-7-oxa-1,2-diaza-benzo[de]anthracen-10-ylmethyl)-guanidine(50)

To the solution of 49 (0.10 g, 0.38 mmol) in DMF (5 mL) was addeddiisopropyl ethylamine (0.83 mmol) and 1H-pyrazole carboxamidinehydrochloride (0.12 g, 0.81 mmol). The reaction mixture was stirred atroom temperature overnight. Diethyl ether was added to form a whiteprecipitate. The precipitate was collected and recrystallized in EtOActo afford a white solid 50 (0.11 g, 83%, HCl salt). MS (ES−): 306;¹H-NMR (DMSO-d₆, 400 MHz): 12.62 (s, 1H), 8.04 (d, J=2.0 Hz, 1H),7.92-7.84 (m, 2H), 7.71 (dd, J=7.5 and 2.0 Hz, 1H), 7.50 (dd, J=8.5 and2.0 Hz, 1H), 7.45 (d, J=8.5 Hz, 1H), 4.47 (s, 2H); Anal. Calcd. forC₁₆H₁₃N₅O₂.1ClH: C, 55.58; H, 4.27; N, 19.76; Cl, 10.38. Found: C,55.94; H, 4.17; N, 19.32; Cl, 10.12.

General Procedure C

To a solution of compound 49 in organic solvents (such as DMF, Dioxane)was added anhydride or acid chloride. The reaction mixture was stirredat various temperature. The solvents was removed in vacuo and theproducts was purified by recrystallization.

2,3-Diacetoxy-N-(3-oxo-2,3-dihydro-7-oxa-1,2-diaza-benzo[de]anthracen-10-ylmethyl)-succinamicacid (51). To the solution of compound 49 (0.20 g, 0.76 mmol) in Dioxane(20 mL) was added diacetyl tartaric anhydride (0.17 g, 0.80 mmol). Thereaction mixture was stirred at room temperature overnight. The solventwas removed in vacuo. The residue was recrystallized in CH₂Cl₂. A whitesolid 51 was afforded in 95% yield. MS: (ES+): 482; ¹H-NMR (DMSO-d₆, 400MHz): 12.66 (s, 1H), 8.86 (d, J=6.2 Hz, 1H), 7.99 (s, 1H), 7.92-7.86 (m,2H), 7.70 (dd, J=7.5 and 2.0 Hz, 1H), 7.40-7.36 (m, 2H), 5.56 (d, J=2.5Hz, 1H), 5.51 (d, J=2.5 Hz, 1H), 4.45 (dd, J=15.0 and 6.3 Hz, 1H), 4.29(dd, J=15.0 and 6.3 Hz, 1H), 2.14 (s, 3H), 1.98 (s, 3H); Anal. Calcd.for C₂₃H₁₉N₃O₉.(1.2H₂O): C, 54.92; H, 4.29; N, 8.35. Found: C, 54.89; H,3.82; N, 8.39.

2,3-Dihydroxy-N-(3-oxo-2,3-dihydro-7-oxa-1,2-diaza-benzo[de]anthracen-10-ylmethyl)-succinamicacid (52). Compound 51 was dissolved in a solution of NaOH (2.5equivalent) in H₂O/Dioxane. The reaction mixture was stirred overnightat 40° C., then was acidified to pH=3. Aqueous layer was extracted withethyl acetate. Organic layer was dried with MgSO₄. Solvent was removedin vacuo to afford a white solid 52 in 25% yield. MS: (ES+): 398; Mp:181-185° C.; ¹H-NMR (DMSO-d₆, 400 MHz): 12.54 (s, 1H), 8.32 (t, J=6.2Hz, 1H), 7.93 (d, J=2.0 Hz, 1H), 7.90-7.80 (m, 2H), 7.62 (dd, J=7.5 and2.0 Hz, 1H), 7.40 (dd, J=8.5 and 2.0 Hz, 1H), 7.25 (d, J=8.5 Hz, 1H),4.30-4.19 (m, 4H); Anal. Calcd for C₁₉H₁₅N₃O₇.(3H₂O): C, 50.56; H, 4.69;N, 9.31. Found: C, 50.59; H, 4.07; N, 9.28.

Pyrrolidine-2-carboxylic acid(3-oxo-2,3-dihydro-7-oxa-1,2-diaza-benzo[de]anthracen-10-ylmethyl)-amide(53). To a solution of compound 49 (0.5 mmol) in DMF was addedtriethylamine (0.6 mmol) and Fmoc-L-Proline chloride (0.6 mmol) slowly.The reaction mixture was stirred at room temperature for 3 hours. Waterwas poured into the reaction mixture. A white precipitate was formed andcollected through filtration. The white solid was recrystallized toafford a white solid in 65% yield. The white solid was dissolved in 20%piperidine in DMF. The solution was stirred at room temperature for 2hours. Water was added to the solution. Aqueous layer was extracted withethyl acetate. Organic layer was collected, washed with brine, anddried. Solvent was removed to afford a white solid 53 in 54% yield. MS(ES+): 363; ¹H-NMR (DMSO-d₆, 400 MHz): 12.64 (s, 1H), 8.59 (t, J=5.8 Hz,1H), 7.98 (d, J=2.0 Hz, 1H), 7.90-7.82 (m, 2H), 7.72 (dd, J=7.5 and 2.0Hz, 1H), 7.43 (dd, J=8.5 and 2.0 Hz, 1H), 7.36 (d, J=8.5 Hz, 1H), 4.35(d, J=3.8 Hz, 2H), 3.62 (m, 1H), 2.98 (t, J=5.2 Hz, 1H), 2.87 (m, 1H),1.99 (m, 1H), 1.73 (m, 1H), 1.64 (m, 2H); Anal. Calcd. forC₂₀H₁₈N₄O₃.(1.2H₂O): C, 62.56; H, 5.35; N, 14.59. Found: C, 62.75; H,5.34; N, 14.52.

(3-Oxo-2,3-dihydro-7-oxa-1,2-diaza-benzo[de]anthracen-10-ylmethyl)-phosphoramidicacid diethyl ester (54). To a solution of compound 49 (0.5 mmol) inanhydrous DMF (20 mL) was added diethyl chlorophosphate (1.5 mmol).After 3 hours solution was concentrated in vacuo, and 50 mL of water wasadded. The aqueous layer was extracted with 30 mL of EtOAc twice.Organic layers were combined and washed with 30 mL of brine. Organiclayer was dried with MgSO₄. Solvent was removed to afford a white solid,which was recrystallized in 10% of EtOAc in hexanes to afford a whitesolid 54 in 50% yield. MS (ES−): 400; Mp: 222.3-224.5° C.; ¹H-NMR(CDCl₃, 400 MHz): 10.21 (s, 1H), 8.10 (d, J=2.5 Hz, 1H), 8.03 (d, J=7.5Hz, 1H), 7.79 (t, J=8.0 Hz, 1H), 7.52 (d, J=8.0 Hz, 1H), 7.47 (dd, J=8.5and 2.0 Hz, 1H), 7.22 (d, J=8.5 Hz, 1H), 4.12 (m, 6H), 1.32 (t, J=6.5Hz, 6H); Anal. Calcd. for C₁₉H₂₀N₃O₅P.(0.3H₂O): C, 56.10; H, 5.10; N,10.33. Found: C, 56.00; H, 5.03; N, 10.13.

4-(4-Dimethylamino-phenylazo)-N-(3-oxo-2,3-dihydro-7-oxa-1,2-diaza-benzo[de]anthracen-10-ylmethyl)-benzenesulfonamide(55). To a solution of compound 49 (0.5 mmol) in anhydrous DMF (20 mL)was added 4-dimethylaminoazobenzene-4′-sulfonyl chloride (0.5 mmol). Themixture was stirred for 3 hours at room temperature. The reactionmixture was concentrated in vacuo, and 50 mL of 50% EtOAc in hexanes wasadded. The precipitate was formed and collected through filtration. Thecrude product was recrystallized in 50% ethanol to afford a orange solid55 in 59% yield. MS (ES+): 553; Mp: >300° C.; ¹H-NMR (DMSO-d₆, 400 MHz):12.63 (s, 1H), 7.90 (d, J=2.0 Hz, 1H), 7.86-7.74 (m, 6H), 7.67 (d, J=9.0Hz, 2H), 7.62 (dd, J=6.6 and 2.5 Hz, 1H), 7.44 (dd, J=8.5 and 2.0 Hz,1H), 7.30 (d, J=8.5 Hz, 1H), 6.81 (d, J=9.0 Hz, 2H), 4.15 (s, 2H), 3.09(s, 6H); Anal. Calcd. for C₂₉H₂₄N₆O₄S.(1.3H₂O): C, 60.47; H, 4.65; N,14.59; S, 5.57. Found: C, 60.72; H, 4.93; N, 15.39; S, 4.93.

EXAMPLE 11 Preparation of diazabenzo[de]anthracen-3-one Derivatives56-61 (Scheme 11)

10-Aminooxymethyl-2H-7-oxa-1,2-diaza-benzo[de]anthracen-3-one (56). To asolution of hydroxy phthalimide (3.6 mmol) in anhydrous DMF was addedsodium anhydride 60% in mineral oil (3.6 mmol). The solution was stirredfor 20 minutes at room temperature. To this solution was added compound41 (3.0 mmol). The reaction mixture was stirred at room temperature fortwo hours and the solvent was removed in vacuo. To the residue was added100 mL of ethyl acetate. The organic layer was washed with water andbrine, then dried over MgSO₄. Ethyl acetate was removed in vacuo. Awhite solid was afforded in 70% yield. The white solid was added to amixture of hydrazine and ethyl alcohol. The mixture was stirred for 2hours under reflux. The solution was concentrated down in vacuo. To theconcentrated solution was added water. A white precipitate was formed,filtered out, and washed with extra water. The white solid was collectedand dried to give a white solid product 56 in 85% yield. MS (ES+): 282;Mp: 294-296° C.; ¹H-NMR (DMSO-d₆, 400 MHz): 12.64 (s, 1H), 8.04 (d,J=2.0 Hz, 1H), 7.92-7.86 (m, 2H), 7.73 (dd, J=7.5 and 2.0 Hz, 1H), 7.50(dd, J=8.5 and 2.0 Hz, 1H), 7.41 (d, J=8.5 Hz, 1H), 4.64 (s, 2H); MS:(ES+): 282; Anal. Calcd. for C₁₅H₁₁N₃O₃: C, 64.05; H, 3.94; N, 14.94.Found: C, 64.24; H, 4.00; N, 14.97.

10-Hydroxymethyl-2H-7-oxa-1,2-diaza-benzo[de]anthracen-3-one (57). To asolution of compound 41 (4.2 mmol) in 20 mL of water and 20 mL ofacetone was added silver nitrate (8.4 mmol). The reaction mixture wasstirred at 50° C. for 2 hours. Black precipitate was filtered out. Thefiltrate was concentrated in vacuo. To the residue was added 100 mL ofethyl acetate. The organic layer was washed with water and brine, thendried with MgSO₄. Solvents was removed in vacuo to afford a white solidin 86% yield. The white solid was added to a mixture of hydrazine andethyl alcohol. The mixture was stirred for 2 hours under reflux. Thesolution was concentrated in vacuo. To the concentrated solution wasadded water. A white precipitate was formed, filtered out, and washedwith extra water. The white solid was collected and dried to give awhite solid product 57 in 95% yield. MS (ES+): 267, ¹H-NMR (DMSO-d6, 400MHz): 12.56 (s, 1H), 7.99 (d, J=2.0 Hz, 1H), 7.91-7.81 (m, 2H), 7.63(dd, J=7.5 and 2.0 Hz, 1H), 7.43 (dd, J=8.5 and 2.0 Hz, 1H), 7.30 (d,J=8.5 Hz, 1H), 4.50 (d, J=6.0 Hz, 2H); Anal. Calcd. for C₁₅H₁₀N₂O₃.(0.08hydrazine): C, 67.02; H, 3.87; N, 11.25. Found: C, 67.29; H, 3.82; N,11.51.

General Procedure D

To a solution of compound 57 in organic solvents (such as DMF, Dioxane)was added anhydride or acid chloride. The reaction mixture was stirredat various temperature. The solvents was removed in vacuo and theproducts was purified by recrystallization.

[2-(3-Oxo-2,3-dihydro-7-oxa-1,2-diaza-benzo[de]anthracen-10-ylmethoxy)-ethyl]-phosphonicacid dibenzyl ester (58). To a solution of dibenzylphosphite (2.6 mmol)in 10 mL of THF was added ethyl acrylate (3.0 mmol), K₂CO₃ (16 mmol),and 0.3 mL of (Bn4NH)₂SO₄ 50% w/w. The reaction mixture was stirred at45° C. overnight. Water was added. The aqueous layer was extracted withethyl acetate. Organic layer was collected and dried with MgSO₄. Solventwas removed to afford a clear oil 3-bis(benzyloxy)phosphorylpropanoicacid in 90% yield. ¹H-NMR (CDCl₃, 400 MHz): 7.33 (m, 10H), 4.90-5.10 (m,4H), 3.61 (s, 3H), 2.50-2.60 (m, 2H), 2.00-2.15 (m, 2H).

To a solution of compound 57 (0.5 mmol) in anhydrous DMF was added3-bis(benzyloxy)phosphorylpropanoic acid (0.75 mmol) and EDC (0.75 mmol)and DMAP (catalytic amount). The reaction mixture was stirred at roomtemperature overnight. Solvent was removed in vacuo. To the residue, 100mL ethyl acetate was added. The organic layer was washed with water, anddried with MgSO₄. Solvent was removed to afford a colorless oil, whichwas recrystallized in EtOAc/Hexanes to afford a white solid 58 in 40%yield. MS (ES−): 581; Mp: 173-175° C. ¹H-NMR (CDCl₃, 400 MHz): 10.01 (s,1H), 8.11 (d, J=2.0 Hz, 1H), 8.07 (d, J=7.5 Hz, 1H), 7.82 (t, J=8.5 Hz,1H), 7.54 (d, J=8.0 Hz, 1H), 7.40 (dd, J=8.5 and 2.0 Hz, 1H), 7.35-7.22(m, 11H), 5.09 (s, 2H), 4.98 (m, 4H), 2.62 (m, 2H), 2.12 (m, 2H); Anal:Calcd for C₃₂H₂₇N₂O₇P: C, 65.98; H, 4.67; N, 4.81. Found: C, 65.69; H,4.82; N, 5.09.

Dimethylamino-acetic acid3-oxo-2,3-dihydro-7-oxa-1,2-diaza-benzo[de]anthracen-10-ylmethyl ester(59). To a solution of compound 57 (0.5 mmol) in anhydrous DMF was addeddimethyl glycine (0.75 mmol) and EDC (0.75 mmol) and DMAP (catalyticamount). The reaction mixture was stirred at room temperature overnight.Solvent was removed in vacuo. To the residue, 100 mL of ethyl acetatewas added. The organic layer was washed with water, and brine, thendried with MgSO₄. The Solvents were removed to afford a colorless oil,which was re-crystallized in EtOAc/Hexanes to afford an off-white solid59 in 30% yield. MS (ES+): 352; MP: 227-230° C.; ¹H-NMR (CDCl₃, 400MHz): 10.15 (s, 1H), 8.14 (d, J=2.0 Hz, 1H), 8.06 (dd, J=8.0 and 1.0 Hz,1H), 7.80 (t, J=8.0 Hz, 1H), 7.53 (dd, J=8.0 and 1.0 Hz, 1H), 7.47 (dd,J=8.5 and 2.0 Hz, 1H), 7.22 (d, J=8.5 Hz, 1H), 5.21 (s, 2H), 3.26 (s,2H), 2.37 (s, 6H); Anal. Calcd. for C₁₉H₁₇N₃O₄: C, 64.95; H, 4.86; N,11.96. Found: C, 64.95; H, 4.78; N, 12.14.

Phosphoric acid dibenzyl ester3-oxo-2,3-dihydro-7-oxa-1,2-diaza-benzo[de]anthracen-10-ylmethyl ester(60). To a solution of compound 57 (0.25 mmol) in anhydrous DMF wasadded dibenzyl phosphate (0.80 mmol) and triphenylphosphine (0.80 mmol).The reaction mixture was stirred while diisopropyl azodicarboxylate wasadded slowly. The reaction mixture was stirred at room temperature for 3hours. Solvent was removed in vacuo. To the residue, 100 mL of ethylacetate was added. The organic layer was washed with water and brine,then dried with MgSO₄. The Solvents were removed to afford a yellow oil.The oil was re-crystallized in EtOAc/Hexanes to afford an off-whitesolid 60 in 40% yield. MS (ES+): 527; Mp: 223-225° C.; ¹H-NMR (CDCl₃,400 MHz): 9.91 (s, 1H), 8.13 (d, J=2.0 Hz, 1H), 8.11 (dd, J=8.0 and 1.0Hz, 1H), 7.85 (t, J=8.0 Hz, 1H), 7.59 (dd, J=8.0 and 1.0 Hz, 1H), 7.45(dd, J=8.5 and 1.0 Hz, 1H), 7.36-7.26 (m, 11H), 5.08 (m, 6H). Anal.Calcd. for C₂₉H₂₃N₂O₆P.(0.15 hydrazine): C, 65.56; H, 4.48; N, 6.06.Found: C, 65.55; H, 4.33; N, 6.09.

Phosphoric acidmono-(3-oxo-2,3-dihydro-7-oxa-1,2-diaza-benzo[de]anthracen-10-ylmethyl)ester(61). To a solution of compound 57 (0.25 mmol) in anhydrous DMF wasadded 1H-tetrazole (2.5 mmol) and di-tert-butylN,N-diethylphosphoramidite (1.25 mmol). The reaction mixture was stirredat room temperature for 2 hours. To the solution was added t-butylhydroperoxide. The reaction mixture was stirred for another one hour.The reaction mixture was cooled to 0° C. and 15 ml of 15% NaHSO₃solution in water was added to the reaction mixture. The mixture wasstirred for another 15 minutes. The mixture was neutralized to pH=8.5and extracted with ethyl acetate. Organic layer was collected, washedwith brine and dried over MgSO₄. The Solvents were removed in vacuo toafford an off-white solid, which was re-crystallized in EtOAc/hexanes togive the intermediate in 78% yield.

The off-white solid was dissolved in 10 mL of CH₂Cl₂ and 4 mL of TFA wasadded to the solution. The reaction mixture was stirred at roomtemperature for 3 hours. Solvent was removed in vacuo. Residue wasre-crystallized in ethyl acetate to afford a yellow solid 61 in 60%yield. MS (ES+): 347; Mp: 246-252° C. ¹H-NMR (DMSO-d₆, 400 MHz): 12.64(s, 1H), 8.09 (s, 1H), 7.93-7.85 (m, 2H), 7.67 (dd, J=7.0 and 2.0 Hz,1H), 7.53 (dd, J=8.5 and 2.0 Hz, 1H), 7.41 (d, J=8.0 Hz, 1H), 4.95 (d,J=7.5 Hz, 2H); Anal. Calcd. for C₁₅H₁₁N₂O₆P: C, 52.04; H, 3.2; N, 8.09.Found: C, 51.76; H, 3.38; N, 8.21.

EXAMPLE 12 Focal Cerebral Ischemia Effect of10-(4-Methyl-piperazin-1-ylmethyl)-2H-7-oxa-1,2-diaza-benzo[de]anthracen-3-one(43)

Rats are allowed free access to water and rat chow (Wayne, Chicago,Ill.) until surgery. Housing and anesthesia concur with guidelinesestablished by the institutional Animal Studies Committee, and are inaccordance with the PHS Guide for the Care and Use of LaboratoryAnimals, USDA Regulations, and the AVMA Panel on Euthanasia guidelines.

The animals are anesthetized with isofluorane (induction, 3%;maintenance, 1.25% in a O₂) through a face mask. During the briefsurgeries, animals were kept warm on a heating blanket. An iv catheteris inserted into the left femoral vein for administration of drugs. Theright middle cerebral artery (MCA) is then exposed by making a verticalskin incision midway between the right eye and ear and the overlyingskull is removed with a dental drill. For transient occlusion, a Codmanmicroclip is applied to the artery at the level of inferior cerebralvein. For permanent occlusion of the MCA, the artery is coagulated atthe level of the inferior cerebral vein with a bipolar cautery unit(Valleylab NS2000, Boulder, Colo.), and cut to prevent spontaneousreperfusion. Both common carotid arteries (CCAs) that had beenpreviously isolated and freed of soft tissues and nerves are thenligated using non-traumatic aneurysm clips. After the wounds are closedwith surgical clips, the animals are allowed to recover from anesthesiaand returned to their cage in a room warmed to 27° C.

For the Pre-Post dosing strategy drugs are first administered as an ivbolus 30 min before MCAO and then 30 min before reperfusion, i.e., onehour post-MCAO. Ninety minutes after the MCAO, the animals are brieflyreanesthetized with isofluorane, and the carotid clips are removed. Theanimal is returned to the warm room overnight.

At 24 hrs after either transient or permanent MCAO, animals are killedwith CO₂ and the heads removed by guillotine. Brains are removed, cooledin ice-cold saline, and sliced into 2 mm coronal sections using a ratbrain matrice (Harvard Bioscience, South Natick, Mass.). The brainslices are incubated in phosphate-buffered saline (pH 7.4) containing 2%TTC at 37° C. for 10 min. and then stored in 10% neutral-bufferedformalin. Cross-sectional area of the TTC-unstained region for eachbrain slice is determined using an image analyzer (MetaMorph, UniversalImaging Corp., West Chester, Pa.). The total volume of infarction in theright hemisphere is calculated by summation of the direct(TTC-negative). The infarcted volumes in vehicle and drug-treated groups(n=8) are tested for significant statistical difference using anunpaired Student-t test (FIG. 1).

EXAMPLE 13 Myocardial Protection of10-(4-Methyl-piperazin-1-ylmethyl)-2H-7-oxa-1,2-diaza-benzo[de]anthracen-3-one(43)

In the present study, we investigated the effects of 43 in the regionalheart ischemia reperfusion model. In regional heart ischemia model,sodium thiopental-anaesthetized male SD rats were subjected to occlusionof LAD coronary artery for 30 min followed by 2 hr of reperfusion. Theinfarct size was quantified by TTC staining method and expressed aspercent of area at risk. Administration of 43 iv. bolus pre-ischemiaplus another iv. bolus post-ischemia exerts significant cardiacprotection in a dose range from 10 to 40 mg/kg.

The reperfusion injury caused by free radicals is not limited tocerebral ischemia. It also contributes significantly to damage of otherorgans including the heart and skeletal muscle. It has been demonstratedthat PARP inhibitors reduce the infarct size caused by ischemia andreperfusion of heart and skeletal muscle of the rabbit. Evidencesuggests that the reperfusion injury for cerebral and myocardialischemia may have a common mechanism in that both injuries are due toPARP activation as a result of DNA damage by free radicals. Transientinhibition of PARP activity is a novel approach for the therapy ofischemia-reperfusion injury of the heart.

The left anterior descending coronary artery of Sprague-Dawley rats wasoccluded for 30 minutes. This was followed by 90-120 minutes of bloodreflow. PARP inhibitor compounds were given intraperitoneally (ip), 60minutes pre-, or intravenously (iv), 15 or 5 minutes pre- and 25 minutespost-onset of ischemia. Infarct size was determined by TTC staining andthe risk area was determined via blue dye injection.

PARP inhibitors exhibited significant reduction in infarct size in thismodel, that is it reduced the infarct volume by between 36%, p=0.001.

EXAMPLE 1410-(4-Methyl-piperazin-1-ylmethyl)-2H-7-oxa-1,2-diaza-benzo[de]anthracen-3-one(43) enhances in vitro sensitivity of melanoma, lymphoma andglioblastoma multiforme cell lines to TMZ

Cell Lines

The murine melanoma cell line B16 of C57BL/6J (H-2^(b)/H-2^(b)) originand the lymphoma cell line L5178Y of DBA/2 (H-2^(d)/H-2^(d)) origin(ATCC, Manassas, Va.) were cultured in RPMI-1640 containing 10% fetalcalf serum (Invitrogen, Milan, Italy), 2 mM L-glutamine, 100 units/mlpenicillin and 100 μg/ml streptomycin (Flow Laboratories, Mc Lean, Va.),at 37° C. in a 5% CO₂ humidified atmosphere. The human glioblastomamultiforme cell line SJGBM2 was cultured in DMEM (Invitrogen)supplemented with 10% fetal calf serum, 2 mM L-glutamine andantibiotics. SJGBM2 cell line was a kind gift from Dr. Peter J. Houghton(St. Jude Children's Research Hospital, Memphis, Tenn., USA).

Drugs

TMZ was provided by Schering-Plough Research Institute (Kenilworth,N.J., USA. Drug stock solutions were prepared by dissolving TMZ indimethyl sulfoxide and 43 in 70 mM PBS without potassium.

In Vitro Studies

Cells were treated with 43 (0.1-20 μM) or with TMZ (1-250 μM). For theexperiments aimed at assessing the enhancing effect of 43 on TMZ-inducedtumor growth inhibition, the methylating agent was added to cellcultures 15 min after 43 was used at concentrations non-toxic, andcapable of abrogating PARP activity. The final concentration of dimethylsulfoxide was always less than 0.1% (v/v) and did not contribute totoxicity (data not shown).

Cells were cultured for three days and apoptosis or cell cycle analysiswere evaluated daily by flow-cytometry analysis of DNA content aspreviously described (Tentori 1997). Briefly, cells were washed with PBSand fixed in 70% ethanol at −20° C. for 18 h. The centrifuged pelletswere resuspended in 1 ml of hypotonic solution containing propidiumiodide (50 μg/ml), 0.1% sodium citrate, 0.1% Triton-X, and RNase (10μg/ml) and incubated in the dark, at 37° C. for 15 min. Data collectionwas gated utilizing forward light scatter and side light scatter toexclude cell debris and aggregates. The propidium iodide fluorescencewas measured on a linear scale using a FACSscan flow cytometer (Bectonand Dickinson, San Jose, Calif., USA). All data were recorded andanalyzed using Cell Quest software. For cell cycle analysis the Mod-fitsoftware was used (Becton and Dickinson).

Long-term survival was analyzed by colony-formation assay. Exponentiallygrowing melanoma and glioblastoma cells were seeded into 6-well platesat a density of 2×10²/well and left to attach for 18 h; cells were thentreated with the compounds under study and cultured to allow colonyformation. After 10 days, colonies (defined as a cluster of >50 cells)were fixed and stained with rhodamine B basic violet 10 (ICN BiomedicalsInc., Aurora, Ohio, USA). Untreated or treated lymphoma cells wereseeded at 1 cell/well in 96-well plates (flat-bottomed) and cultured.After 10 days, colonies were counted to determine cloning efficiency,using an optical microscope (Tentori 2001b).

Cell line chemosensitivity to TMZ, 43 or to the drug combination wasevaluated in terms of IC₅₀, i.e. the concentration of the drug expressedin μM, capable of inhibiting colony-forming ability by 50%. The IC₅₀ wascalculated on the regression line in which the number of colonies wasplotted vs the drug concentration.

In Vivo Studies

The intracranial transplantation procedure was performed as previouslydescribed (Tentori 2002b). Cells (10⁴ in 0.03 ml of RPMI-1640) wereinjected intra-cranially (ic) through the center-middle area of thefrontal bone to a 2 mm depth, using a 0.1 ml glass microsyringe and a27-gauge disposable needle.

Murine melanoma B16 or lymphoma L5178Y cells (10⁴) were injected ic intomale B6D2F1 (C57BL/6 ×DBA/2) mice. Human glioblastoma multiforme SJGBM2cells (10⁶) were injected ic into male athymic BALB/c mice (nu/nugenotype). Before tumor challenge, animals were anesthetized withketamine (100 mg/kg) and xylazine (5 mg/kg) in 0.9% NaCl solution (10ml/kg/ip).

Histological evaluation of tumor growth in the brain was performed 1-5days after tumor challenge, in order to select the timing of treatment.Brains were fixed in 10% phosphate-buffered formaldehyde, cut along theaxial plane and embedded in paraffin. Histological sections (5 μm thick)were stained with hematoxylin-eosin and analyzed by light microscopy.

Drug toxicity was evaluated by treating intact mice (5/group) with thecompounds under study, used as single agents or in combination. Controlgroups were treated with vehicles only. Body weight was measured threetimes weekly and survivals were recorded for 3 weeks after the lasttreatment. Toxicity was assessed on the basis of apparent drug-relateddeaths and net body weight loss [i.e., (initial weight−lowestweight/initial weight)×100%]. Death was considered drug-related whenoccurring within 7 days after the last treatment.

43 was dissolved in 70 mM PBS without potassium, and injectedintravenously (iv) at different doses (40-200 mg/kg) to establish themaximal tolerated dose.

TMZ was dissolved in dimethyl-sulfoxide (40 mg/ml), diluted in saline (5mg/ml) and administered ip at doses commonly used for in vivopreclinical studies (Friedman 1995; Middleton 2000; Kokkinakis 2001).Experiments were performed using different doses and schedules of TMZ+43to determine the maximal tolerated dose of the drug combination. 43 wasadministered 15 min before TMZ administration. Control mice were alwaysinjected with drug vehicles.

In tumor-bearing mice treatment was started on day 2 after challenge,when tumor infiltration in the surrounding brain tissue washistologically evident. Since the antitumor activity of TMZ and PARPinhibitors can be improved by fractionated modality of treatment(Tentori 2002b), the maximal tolerated dose of the drug combination wasdivided into daily administrations of 100 mg/kg TMZ+40 mg/kg 43 for 3days.

Mice were monitored for mortality for 90 days. Median survival times(MST) were determined and the percentage of increase in lifespan (ILS)was calculated as: {[MST (days) of treated mice/MST (days) of controlmice]−1}×100. Efficacy of treatments was evaluated by comparing survivalcurves between treated and control groups.

To assess the ability of different treatments to reduce tumor growth,histological examination of the brains was performed using additionalanimals that were not considered for the analysis of survival. Mice weresacrificed at different time points after tumor challenge, selectedwithin the MST range of untreated tumor-bearing animals.

The efficacy of TMZ±43 treatment was also evaluated on melanoma growingsubcutaneously (sc). For this purpose B16 cells (2.5×10⁵) wereinoculated sc in the flank of the animal. Tumors were measured withcalipers and volume calculated according to the formula:[(width)²×length]/2. Treatment was started 6 days after challenge, whenthe volume of tumor nodules reached 100-150 mm³. Melanoma growth wasmonitored, by measuring tumor nodules every 3 days for 3 weeks.

To evaluate the influence of the drugs under study on generation ofartificial metastases, B16 cells (2.5×10⁵ in 0.02 ml) were injected intothe tail vein of B6D2F1 mice. Animals were treated with the drugs understudy using the 3-days schedule (see above). Two weeks after tumorchallenge, animals were sacrificed, lungs removed and fixed in Bouin'ssolution to distinguish tumor nodules from lung tissue. The number ofmetastases was determined using a dissecting microscope.

All procedures involving mice and care were performed in compliance withnational and international guidelines (European Economy CommunityCouncil Directive 86/109, OLJ318, Dec. 1, 1987 and NIH Guide for careand use of laboratory animals, 1985.)

Statistical Analysis

Survival curves were generated by Kaplan-Meier product-limit estimateand statistical differences between the various groups were evaluated bylog-rank analysis (software Primer of Biostatistics, McGraw-Hill, NewYork, N.Y.). For statistical analysis of tumor growth or metastasisnumber, the significance of the differences between experimental groupswas evaluated by t test. Ps are two-sided (software Microsoft excel).

Initially B16, SJGBM2 and L5178Y cells were exposed to 1-25 μM 43, assingle agent. Cell growth was analysed by colony formation assay and theresults indicated that 43 exhibited some intrinsic growth inhibition andthat B16 melanoma was more susceptible to the antiproliferative effectinduced by 43 with respect to SJGBM2 and L5178Y cell lines.

For each cell line 43 concentrations devoid of toxic effects (0.3-1.2μM) were tested for their ability to enhance growth inhibition inducedby TMZ. In all tumor cell lines, the PARP inhibitor increased growthinhibition induced by TMZ. In the case of B16 cell line, the maximalenhancement of TMZ-induced growth inhibition was achieved at 0.6 μMconcentration (10-fold). In B16 cells, 43 at the concentration of 1.2 μMinduced 18±3% growth inhibition with respect to control and was notconsidered for combination studies with TMZ. For SJGBM2 and L5178Y celllines, the maximal increase of TMZ growth inhibitory effect was observedat 1.2 μM 43 (˜4-fold for SJGBM2 and 10-fold for L5178Y, respectively).In SJGBM2 and L5178Y cell lines 2.5 μM 43 showed intrinsic growthinhibitory effect, therefore this concentration was not tested inassociation with TMZ.

In B16 and SJGBM2 cells TMZ+43 mainly provoked cytostasis withoutinduction of apoptosis, while in L5178Y lymphoma cells the drugcombination induced also apoptosis (data not shown).

EXAMPLE 15 Systemic administration of10-(4-Methyl-piperazin-1-ylmethyl)-2H-7-oxa-1,2-diaza-benzo[de]anthracen-3-one(43)+TMZ enhances survival of mice bearing melanoma, lymphoma orglioblastoma multiforme at the CNS site

The antitumor activity of the drug combination, at the highest tolerabledose (TMZ 100 mg/kg/day ip+43 40 mg/kg/day iv, for 3 days), wasinitially tested in B16 melanoma growing subcutaneously in B6D2F1 miceand compared to the effects induced by TMZ or 43 used as single agents.The results show that 43 significantly enhanced (P<0.0001) the antitumoreffects of TMZ, while treatment with 43 only, did not affect tumorgrowth.

The same drug schedule was used to investigate whether systemicadministration of 43, which is capable of crossing the blood-brainbarrier, might increase efficacy of TMZ also against B16 melanomagrowing at the CNS site. Drug treatment was started 2 days after tumorchallenge, when neoplastic infiltration of the brain tissue wasevidenced in histological sections. The results show that the increasein survival time induced by 43+TMZ combination was significantly higher(P<0.0001) than that provoked by TMZ as single agent. The increase insurvival detected in the group treated with the drug combination wasaccompanied by a marked reduction of tumor growth. Histological studiesrevealed a pronounced and diffuse tumor infiltration in the brain ofcontrol or 43 treated mice, limited but multifocal infiltration inTMZ-treated mice, whereas only few infiltrating melanoma cells in 43+TMZtreated animals.

The PARP inhibitor 43 also increased the anti-metastatic activity of TMZagainst B16 melanoma. In fact, the number of pulmonary metastasesobserved after treatment with 43+TMZ was significantly lower (P=0.004)than that detected in mice treated with TMZ used as single agent.

Systemic administration of 43+TMZ significantly increased survival ofB6D2F1 mice bearing L5178Y lymphoma growing in the brain. The increasein median survival time induced by the drug combination wassignificantly higher than that provoked by TMZ used as single agent.

The efficacy of drug treatment was then investigated using an orthotopicmodel of a human glioblastoma multiforme xenograft in nude mice. Theresults indicate that systemic administration of 43+TMZ significantlyprolonged survival of tumor bearing mice with respect to controls or toanimals treated with the single agents. It should be noted that in thistumor model TMZ was completely ineffective.

Microscopic examination of control animals injected with SJGBM2 revealedmultifocal brain involvement. Treatment with 43+TMZ resulted in adecreased number of sites of neoplastic infiltration. In the controlgroup, all animals presented tumor infiltration in at least two brainregions distant from the site of injection, whereas in the group treatedwith the drug combination only 2/7 mice showed this pattern (total brainregions infiltrated by tumor cells: control, 24; 43+TMZ: 13; P=0.0007).Moreover, brains of control mice showed large tumor masses both at thesite of injection and in the parenchyma surrounding the ventricles; incontrast, animals treated with 43+TMZ showed minimal tumor infiltration.

EXAMPLE 16 Measuring Altered Gene Expression in mRNA Senescent Cells

Gene expression alteration may be measured with human fibroblast BJcells which, at Population Doubling (PDL) 94, are plated in regulargrowth medium and then changed to low serum medium to reflectphysiological conditions described in Linskens, et al., Nucleic AcidsRes. 23:16:3244-3251 (1995). A medium of DMEM/199 supplemented with 0.5%bovine calf serum is used. The cells are treated daily for 13 days. Thecontrol cells are treated with and without the solvent used toadminister the PARP inhibitor. The untreated old and young control cellsare tested for comparison. RNA is prepared from the treated and controlcells according to the techniques described in PCT Publication No.96/13610 and Northern blotting is conducted. Probes specific forsenescence-related genes are analyzed, and treated and control cellscompared. In analyzing the results, the lowest level of gene expressionis arbitrarily set at 1 to provide a basis for comparison. Three genesparticularly relevant to age-related changes in the skin are collagen,collagenase and elastin. West, Arch. Derm. 130:87-95 (1994). Elastinexpression of the cells treated with the PARP inhibitor is expected tobe significantly increased in comparison with the control cells. Elastinexpression should be significantly higher in young cells compared tosenescent cells, and thus treatment with the PARP inhibitor should causeelastin expression levels in senescent cells to change to levels similarto those found in much younger cells. Similarly, a beneficial effectshould be seen in collagenase and collagen expression with treatmentwith the PARP inhibitors.

EXAMPLE 17 Measuring Altered Gene Expression of Protein in SenescentCells

Gene expression alteration may be measured with approximately 105 BJcells, at PDL 95-100 which are plated and grown in 15 cm dishes. Thegrowth medium is DMEM/199 supplemented with 10% bovice calf serum. Thecells are treated daily for 24 hours with the PARP inhibitors of (100μg/1 mL of medium) WO 99/11645. The cells are washed with phosphatebuffered solution (PBS), then permeablized with 4% paraformaldehyde for5 minutes, then washed with PBS, and treated with 100% cold methanol for10 minutes. The methanol is removed and the cells are washed with PBS,and then treated with 10% serum to block nonspecific antibody binding.About 1 mL of the appropriate commercially available antibody solutions(1:500 dilution. Vector) is added to the cells and the mixture incubatedfor 1 hour. The cells are rinsed and washed three times with PBS. Asecondary antibody, goat anti-mouse IgG (1 mL) with a biotin tag isadded along with 1 mL of a solution containing streptavidin conjugatedto alkaline phosphatase and 1 mL of NBT reagent (Vector). The cells arewashed and changes in gene expression are noted calorimetrically. Foursenescence-specific genes—collagen I, collagen III, collagenase, andinterferon gamma—in senescent cells treated with the PARP inhibitor aremonitored and the results should show a decrease in interferon gammaexpression with no observable change in the expression levels of theother three gens, demonstrating that the PARP inhibitors can altersenescence-specific gene expression.

EXAMPLE 18 Extending or Increasing Proliferative Capacity and Lifespanof Cells

To demonstrate the effectiveness of the present method for extending theproliferative capacity and lifespan of cells, human fibroblast cellslines (either W138 at Population Doubling (PDL) 23 or BJ cells at PDL71) are thawed and plated on T75 flasks and allowed to grow in normalmedium (DMEM/M199 plus 10% bovine calf serum) for about a week, at whichtime the cells are confluent, and the cultures are therefor ready to besubdivided. At the time of subdivision, the media is aspirated, and thecells rinsed with phosphate buffer saline (PBS) and then trypsinized.The cells are counted with a Coulter counter and plated at a density of10⁵ cells per cm in 6-well tissue culture plates in DMEM/199 mediumsupplemented with 10% bovine calf serum and varying amounts (0.10 μM,and 1 mM: from a 100× stock solution in DMEM/M199 medium) of a PARPinhibitor. This process is repeated every 7 days until the cells appearto stop dividing. The untreated (control) cells reach senescence andstop dividing after about 40 days in culture. Treatment of cells with 10μM 3-AB appears to have little or no effect in contrast to treatmentwith 100 μM 3-AB which appears lengthen the lifespan of the cells andtreatment with 1 mM 3-AB which dramatically increases the lifespan andproliferative capacity of the cells. The cells treated with 1 mM 3-ABwill still divide after 60 days in culture.

EXAMPLE 19 Neuroprotective Effects of PARP Inhibitors on ChronicConstriction Injury (CCI) in Rats

Adult male Sprague-Dawley rats, 300-350 g, are anesthetized withintraperitoneal 50 mg/kg sodium pentobarbital. Nerve ligation isperformed by exposing one side of the rat's sciatic nerves anddissecting a 5-7 mm-long nerve segment and closing with four looseligatures at a 1.0-1.5-mm, followed by implanting of an intrathecalcatheter and inserting of a gentamicin sulfate-flushed polyethylene(PE-10) tube into the subarachnoid space through an incision at thecisterna magna. The caudal end of the catheter is gently threaded to thelumbar enlargement and the rostral end is secured with dental cement toa screw embedded in the skull and the skin wound is closed with woundclips.

Thermal hyperalgesia to radiant heat is assessed by using apaw-withdrawal test. The rat is placed in a plastic cylinder on a 3-mmthick glass plate with a radiant heat source from a projection bulbplaced directly under the plantar surface of the rat's hindpaw. Thepaw-withdrawal latency is defined as the time elapsed from the onset ofradiant heat stimulation to withdrawal of the rat's hindpaw.

Mechanical hyperalgesia is assessed by placing the rat in a cage with abottom made of perforated metal sheet with many small square holes.Duration of paw-withdrawal is recorded after pricking the mid-plantarsurface of the rat's hindpaw with the tip of a safety pin insertedthrough the cage bottom.

Mechano-allodynia is assessed by placing a rat in a cage similar to theprevious test, and applying von Frey filaments in ascending order ofbending force ranging from 0.07 to 76 g to the mid-plantar surface ofthe rat's hindpaw. A von Frey filament is applied perpendicular to theskin and depressed slowly until it bends. A threshold force of responseis defined as the first filament in the series to evoke at least oneclear paw-withdrawal out of five applications.

Dark neurons are observed bilaterally within the spinal cord dorsalhorn, particularly in laminae I-II, of rats 8 days after unilateralsciatic nerve ligation as compared with sham operated rats. Variousdoses of PARP inhibitors are tested in this model and shown to reduceboth incidence of dark neurons and neuropathic pain behavior in CCIrats.

The invention being thus described, it will be obvious that the same maybe varied in many ways. Such variations are not to be regarded as adeparture from the spirit and scope of the invention, and all suchmodifications are intended to be included within the scope of thefollowing claims. All references cited herein are incorporated in theirentirety by reference herein.

1. A method of treating cancer in a mammal, wherein the cancer isselected from the group consisting of melanoma, lymphoma, andglioblastoma multiforme, comprising administering to the mammal aneffective amount of temozolomide and a compound selected from the groupconsisting of:


2. A method of claim 1, wherein the cancer is glioblastoma multiforme.